Polyhydroxyalkanoates of narrow molecular weight distribution prepared in transgenic plants

ABSTRACT

Methods for the biosynthesis of polyhydroxyalkanoate homopolymers and copolymers are described. In a preferred embodiment, the polymers have a single mode molecular weight distribution, and more preferably have a distribution of between about 2 and about 4, and most preferably about 2.1 or 2.5.

RELATED U.S. APPLICATION DATA

This is a divisional of co-pending application Ser. No. 08/912,205,filed Aug. 15, 1997, which is a continuation-in-part of application Ser.No. 673,388, filed Jun. 25, 1996 (now U.S. Pat. No. 5,958,745), which isa continuation-in-part of application Ser. No. 628,039, filed Apr. 4,1996, (now U.S. Pat. No. 5,942,660), which is a continuation-in-part ofapplication Ser. No. 614,877, filed Mar. 3, 1996 (now U.S. Pat. No.5,959,179).

FIELD OF INVENTION

The present invention relates to genetically engineered plants andbacteria. In particular, it relates to methods for optimizing substratepools to facilitate the biosynthetic production of commercially usefullevels of polyhydroxyalkanoates (PHAs) in bacteria and plants.

BACKGROUND OF INVENTION

PHAs are bacterial polyesters that accumulate in a wide variety ofbacteria. These polymers have properties ranging from stiff and brittleplastics to rubber-like materials, and are biodegradable. Because ofthese properties, PHAs are an attractive source of nonpolluting plasticsand elastomers.

The present invention especially relates to the production ofcopolyesters of β-hydroxybutyrate (3HB) and β-hydroxyvalerate (3HV),designated P(3HB-co-3HV) copolymer, and derivatives thereof.

Currently, there are approximately a dozen biodegradable plastics incommercial use that possess properties suitable for producing a numberof specialty and commodity products (Lindsay, 1992). One suchbiodegradable plastic in the polyhydroxyalkanoate (PHA) family that iscommercially important is Biopol™, a random copolymer of3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV). This bioplastic isused to produce biodegradable molded material (e.g., bottles), films,coatings, and in drug release applications. Biopol™ is produced via afermentation process employing the bacterium Alcaigenes eutrophus(Byrom, 1987). The current market price is $6-7/lb, and the annualproduction is 1,000 tons. By best estimates, this price can be reducedonly about 2-fold via fermentation (Poirier et al., 1995). Competitivesynthetic plastics such as polypropylene and polyethylene cost about35-45¢/lb (Layman, 1994). The annual global demand for polyethylenealone is about 37 million metric tons (Poirier et al., 1995). It istherefore likely that the cost of producing P(3HB-co-3HV) by microbialfermentation will restrict its use to low-volume specialty applications.

Nakamura et al. (1992) reported using threonine (20 g/L) as the solecarbon source for the production of P(3HB-co-3HV) copolymer in A.eutrophus. These workers initially suggested that the copolymer mightform via the degradation of threonine by threonine deaminase, withconversion of the resultant α-ketobutyrate to propionyl-CoA. However,they ultimately concluded that threonine was utilized directly, withoutbreaking carbon-carbon bonds, to form valeryl-CoA as the 3HV precursor.The nature of this chemical conversion was not described, but since thebreaking of carbon-carbon bonds was not postulated to occur, the pathwaycould not involve threonine deaminase in conjunction with an α-ketoaciddecarboxylating step to form propionate or propionyl-CoA. In theexperiments of Nakamura et al, the PHA polymer content was very low (<6%of dry cell weight). This result, in conjunction with the expense offeeding bacteria threonine, makes their approach impractical for thecommercial production of P(3HB-co3HV) copolymer.

Yoon et al. (1995) have shown that growth of Alcaligenes sp. SH-69 on amedium supplemented with threonine, isoleucine, or valine resulted insignificant increases in the 3HV fraction of the P(3HB-co-3HV)copolymer. In addition to these amino acids, glucose (3% wt/vol) wasalso added to the growth media. In contrast to the results obtained byNakamura et al. (1992), growth of A. eutrophus under the conditionsdescribed by Yoon et al. (1995) did not result in the production ofP(3HB-co-3HV) copolymer when the medium was supplemented with threonineas the sole carbon source. From their results, Yoon et al. (1995)implied that the synthetic pathway for the 3HV component inP(3HB-co-3HV) copolymer is likely the same as that described in WO91/18995 and Steinbüchel and Pieper (1992). This postulated syntheticpathway involves the degradation of isoleucine to propionyl-CoA (FIG.3).

U.S. Pat. No. 5,602,321 teaches the insertion and expression of polymerbiosynthesis genes in plants, and preferably in cotton. Geneticconstructs encoding ketothiolase, acetoacetyl CoA reductase, and PHBsynthase enzymes were introduced into cotton.

Sim et al. (1997) reported that the amount of polyhydroxyalkanoatesynthase protein in a bacterial cell affected the molecular weight andpolydispersity of polymers produced therein. Increased synthaseconcentrations led to the biosynthesis of lower molecular weightpolymer.

John and Keller (1996) obtained polyhydroxybutyrate from cottontransformed with the phaB and phaC genes from Alcaligenes eutrophus. Amajor fraction of the polymer obtained had a molecular weight in excessof 600,000. Polydispersity of the polymer is not discussed, nor issufficient data presented to allow calculation thereof.

Poirier et al. (1995b) described the biosynthesis of polyhydroxybutyratein a suspension culture of Arabidopsis thaliana plant cells expressingthe phbB and phbC genes from Alcaligenes eutrophus. No C3 or C53-hydroxyacids were detected by gas chromatography of plant extracts.The polyhydroxybutyrate was found to have a broad molecular weightdistribution of 10.5. Unlike bacterially produced polymer, the plantcell produced material displayed a polymodal distribution, comprising atleast three distinct subpopulations of molecular weight ranges.

The PHB Biosynthetic Pathway

Polyhydroxybutyrate (PHB) was first discovered in 1926 as a constituentof the bacterium Bacillus megaterium (Lemoigne, 1926). Since then, PHAssuch as PHB have been found in more than 90 different genera ofgram-negative and gram-positive bacteria (Steinbüchel, 1991). Thesemicroorganisms produce PHAs using R-β-hydroxyacyl-CoAs as the directmetabolic substrate for a PHA synthase, and produce polymers ofR-(3)-bydroxyalkanoates having chain lengths ranging from C3-C14(Steinbüchel and Valentin, 1995).

To date, the best understood biochemical pathway for PHB production isthat found in the bacterium Alcaligenes eutrophus (Dawes and Senior,1973; Slater et al., 1988; Schubert et al., 1988; Peoples and Sinskey,1989a and 1989b). This pathway, which is also utilized by othermicroorganisms, is summarized in FIG. 1. In this organism, an operonencoding three gene products, i.e., PHB synthase, β-ketothiolase, andacetoacetyl-CoA reductase, encoded by the phbC, phbA, and phbB genes,respectively, are required to produce the PHA homopolymerR-polyhydroxybutyrate (PHB).

As further shown in FIG. 1, acetyl-CoA is the starting substrateemployed in the biosynthetic pathway. This metabolite is naturallyavailable for PHB production in plants and bacteria when these organismsare genetically manipulated to produce the PHB polyester.

Recently, a multi-enzyme pathway was successfully introduced into plantsfor the generation of polyhydroxybutyrate (PHB) (Poirier et al., 1992).

The P(3HB-co-3HV) Copolymer Biosynthetic Pathway

As noted above, P(3HB-co-3HV) random copolymer, commercially known asBiopol™, is produced by fermentation employing A. eutrophus. A proposedbiosynthetic pathway for P(3HB-co-3HV) copolymer production is shown inFIG. 2. Production of this polymer in plants has not yet beendemonstrated.

The successful production of P(3HB-co-3HV) copolymer in plants orbacteria requires the generation of substrates that can be utilized bythe PHA biosynthetic enzymes. For the 3HB component of the polymer, thesubstrate naturally exists in plants in sufficient concentration in theform of acetyl-CoA (Nawrath et al., 1994). This is not true for the 3HVcomponent of the copolymer, however. In this case, the startingsubstrate is propionyl-CoA. The presence of sufficient pools ofacetyl-CoA and propionyl-CoA in plants and microorganisms, along withthe proper PHA biosynthetic enzymes (i.e., a β-ketothiolase, aβ-ketoacyl-CoA reductase, and a PHA synthase), would make it possible toproduce copolyesters of P(3HB-co-3HV) in these organisms.

The present invention provides a variety of different methods foroptimizing levels of substrates employed in the biosynthesis ofcopolymers of 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV) inplants and bacteria via manipulation of normal metabolic pathways usingrecombinant DNA techniques.

In one aspect, the present invention provides methods for the productionof enhanced levels of threonine, α-ketobutyrate, propionyl-CoA,β-ketovaleryl-CoA, and β-hydroxyvaleryl-CoA, all of which aremetabolites in the biosynthetic pathway of 3HV in the P(3HB-co-3HV)copolymer, from various carbon sources in plants or bacteria. This isachieved by providing a variety of wild-type and/or deregulated enzymesinvolved in the biosynthesis of the aspartate family of amino acids(i.e., aspartate, threonine, lysine, and methionine), and wild-type orderegulated forms of enzymes involved in the conversion of threonine toP(3HB-co-3HV) copolymer endproduct. Using these enzymes, the levels ofthe above-noted metabolites can be increased in plants and bacteria inthe range of from about 1-10 fold, 1-100 fold, or 1-1000 fold.

In another aspect, the present invention provides methods for thebiological production of P(3HB-co-3HV) copolymers in plants and bacteriautilizing propionyl-CoA as a substrate. As shown in FIG. 3,propionyl-CoA can be produced through a variety of engineered metabolicpathways. Introduction into plants and bacteria of appropriateβ-ketothiolases capable of condensing acetyl-CoA with itself and/or withpropionyl-CoA, along with appropriate β-ketoacyl-CoA reductases and PHAsynthases, in combination with various enzymes involved in asparatefamily amino acid biosynthesis and the conversion of threonine to PHAcopolymer precursors, will permit these organisms to produceP(3HB-co-3HV) copolymers. The PHA biosynthetic enzymes can be those ofA. eutrophus or other organisms, or enzymes that catalyze reactionsinvolved in fatty acid biosynthesis or degradation, ultimately resultingin the conversion of acetyl-CoA and propionyl-CoA to P(3HB-co-3HV). Inplants, these enzymes can be expressed in the cytoplasm or targeted toorganelles such as plastids (e.g., those of leaves or seeds) ormitochondria via the use of transit peptides for enhanced production ofpolyhydroxyalkanoates. Alternatively, plastids can be transformed withrecombinant constructs that facilitate expression of these enzymesdirectly within the plastids themselves.

The enzymes discussed herein can be employed alone or in variouscombinations in order to enhance the levels of threonine,α-ketobutyrate, propionate, propionyl-CoA, β-ketovaleryl-CoA, andβ-hydroxyvaleryl-CoA, and for the production of P(3HB-co-3HV) copolymer.

More specifically, the present invention encompasses the followingaspects:

An isoleucine-deregulated mutein of E. coli threonine deaminase, whereinleucine at amino acid position 447 is replaced with an amino acidselected from the group consisting of alanine, isoleucine, valine,proline, phenylalanine, tryptophan, and methionine. In one aspect,leucine at amino acid position 447 can be replaced with phenylalanine.

An isoleucine-deregulated mutein of E. coli threonine deaminase, whereinleucine at amino acid position 447 is replaced with an amino acidselected from the group consisting of alanine, isoleucine, valine,proline, phenylalanine, tryptophan, and methionine, and leucine at aminoacid position 481 is replaced with an amino acid selected from the groupconsisting of alanine, isoleucine, valine, proline, phenylalanine,tryptophan, and methionine. In one aspect, leucine at amino acidposition 447 can be replaced with phenylalanine, and leucine at aminoacid position 481 can be replaced with phenylalanine.

An α-ketoacid dehydrogenase complex, comprising an α-ketoaciddecarboxylase E1 subunit, a dihydrolipoyl transacylase E2 subunit, and adihydrolipoyl dehydrogenase E3 subunit, wherein said α-ketoaciddecarboxylase E1 subunit exhibits improved binding and decarboxylatingproperties with α-ketobutyrate compared to pyruvate decarboxylase E1subunit naturally present in a host cell pyruvate dehydrogenase complex.The α-ketoacid decarboxylase E1 subunit can be a branched-chainα-ketoacid decarboxylase E1 subunit such as bovine kidney, Pseudomonasputida, or Bacillus subtilis branched-chain α-ketoacid dehydrogenase E1subunit.

An α-ketoacid dehydrogenase complex, comprising an α-ketoaciddecarboxylase E1 subunit, a dihydrolipoyl transacylase E2 subunit, and adihydrolipoyl dehydrogenase E3 subunit, wherein said E1 and E2 subunitsexhibit improved binding/decarboxylating and transacylase properties,respectively, with α-ketobutyrate compared with pyruvate decarboxylaseE1 subunit and dihydrolipoyl transacetylase E2 subunit, respectively,naturally present in a host cell pyruvate dehydrogenase complex. Theα-ketoacid decarboxylase E1 and dihydrolipoyl transacylase E2 subunitscan be branched-chain α-ketoacid dehydrogenase E1 and E2 subunits suchas those from bovine kidney, Pseudomonas putida, or Bacillus subtilis.

An isolated β-ketothiolase capable of condensing acetyl-CoA andpropionyl-CoA to produce β-ketovaleryl-CoA.

An isolated β-ketothiolase capable of condensing acetyl-CoA andbutyryl-CoA to produce β-ketocaproyl-CoA.

An isolated β-ketothiolase capable of:

condensing two molecules of acetyl-CoA to produce acetoacetyl-CoA;

condensing acetyl-CoA and propionyl-CoA to produce β-ketovaleryl-CoA;and

condensing acetyl-CoA and butyryl-CoA to produce β-ketcaproyl-CoA.

The foregoing isolated β-ketothiolase is exemplified by BktBβ-keto-thiolase having the amino acid sequence shown in SEQ ID NO:11.

Isolated DNA molecules comprising a nucleotide sequence encoding theisoleucine-deregulated muteins of E. coli threonine deaminase describedherein.

An isolated DNA molecule comprising the nucleotide sequence shown in SEQID NO:5.

An isolated DNA molecule comprising the nucleotide sequence shown in SEQID NO:8.

An isolated DNA molecule comprising a nucleotide sequence selected fromthe group consisting of:

(a) the nucleotide sequence shown in SEQ ID NO:9 or the complementthereof;

(b) a nucleotide sequence that hybridizes to said nucleotide sequence of(a) under a wash stringency equivalent to 0.5×SSC to 2×SSC, 0.1% SDS, at55-65° C., and which encodes an enzyme having enzymatic activity similarto that of A. eutrophus BktB β-ketothiolase;

(c) a nucleotide sequence encoding the same genetic information as saidnucleotide sequence of (a), but which is degenerate in accordance withthe degeneracy of the genetic code; and

(d) a nucleotide sequence encoding the same genetic information as saidnucleotide sequence of (b), but which is degenerate in accordance withthe degeneracy of the genetic code.

The foregoing isolated DNA molecule, wherein said wash stringency isequivalent to 2×SSC, 0.1% SDS, at 55° C.

The foregoing isolated DNA molecule, wherein said wash stringency isequivalent to 1×SSC, 0.1% SDS, at 55° C.

The foregoing isolated DNA molecule, wherein said wash stringency isequivalent to 0.5×SSC, 0.1% SDS, at 55° C.

An isolated DNA molecule, comprising the nucleotide sequence shown inSEQ ID NO:9 or the complement thereof.

A recombinant vector, comprising any of the foregoing nucleotidesequences operatively linked to a promoter and 5′ and 3′ regulatorysequences sufficient to drive expression of said nucleotide sequences ina host cell.

A recombinant vector, comprising a nucleotide sequence encoding E. colithreonine deaminase wherein leucine at position 481 is replaced withphenylalanine, operatively linked to a promoter and 5′ and 3′ regulatorysequences sufficient to drive expression of said nucleotide sequence ina host cell.

The foregoing recombinant vector, wherein said nucleotide sequencecomprises the sequence shown in SEQ ID NO:7.

A host cell, comprising any of the foregoing recombinant vectors.

A host cell, comprising an α-ketoacid dehydrogenase complex comprisingan α-ketoacid decarboxylase E1 subunit, a dihydrolipoyl transacylase E2subunit, and a dihydrolipoyl dehydrogenase E3 subunit, wherein saidα-ketoacid decarboxylase E1 subunit exhibits improved binding anddecarboxylating properties with α-ketobutyrate compared to pyruvatedecarboxylase E1 subunit naturally present in a host cell pyruvatedehydrogenase complex, wherein said host cell produces an increasedamount of propionyl-CoA compared to a corresponding host cell comprisingwild-type pyruvate dehydrogenase complex. The α-ketoacid decarboxylaseE1 subunit which exhibits improved binding and decarboxylatingproperties with α-ketobutyrate can be a branched-chain α-ketoaciddecarboxylase E1 subunit.

A host cell, comprising an α-ketoacid dehydrogenase complex, comprisingan α-ketoacid decarboxylase E1 subunit, a dihydrolipoyl transacylase E2subunit, and a dihydrolipoyl dehydrogenase E3 subunit, wherein said E1and E2 subunits exhibit improved binding/decarboxylating andtransacylase properties, respectively, with α-ketobutyrate compared withpyruvate decarboxylase E1 subunit and dihydrolipoyl transacetylase E2subunit, respectively, naturally present in a host cell pyruvatedehydrogenase complex, wherein said host cell produces an increasedamount of propionyl-CoA compared to a corresponding host cell comprisingwild-type pyruvate dehydrogenase complex.

A plant, the genome of which comprises introduced DNA encoding awild-type or deregulated aspartate kinase enzyme;

wherein said introduced DNA is operatively linked to regulatory signalsthat cause expression of said introduced DNA; and

wherein cells of said plant produce an elevated amount of threoninecompared to that in cells of a corresponding, wild-type plant notcomprising said introduced DNA.

A plant, the genome of which comprises introduced DNAs encoding thefollowing enzymes:

a wild-type or deregulated aspartate kinase enzyme; and

threonine synthase;

wherein said introduced DNAs are operatively linked to regulatorysignals that cause expression of said introduced DNAs; and

wherein cells of said plant produce an elevated amount of threoninecompared to that in cells of a corresponding, wild-type plant notcomprising said introduced DNAs.

A plant, the genome of which comprises introduced DNAs encoding thefollowing enzymes:

a wild-type or deregulated aspartate kinase; and

homoserine dehydrogenase;

wherein each of said introduced DNAs is operatively linked to regulatorysignals that cause expression of said introduced DNAs; and

wherein cells of said plant produce an elevated amount of threoninecompared to that in cells of a corresponding, wild-type plant notcomprising said introduced DNAs.

A plant, the genome of which comprises introduced DNAs encoding thefollowing enzymes:

a wild-type or deregulated aspartate kinase;

homoserine dehydrogenase; and

threonine synthase;

wherein each of said introduced DNAs is operatively linked to regulatorysignals that cause expression of said introduced DNAs; and

wherein cells of said plant produce an elevated amount of threoninecompared to that in cells of a corresponding, wild-type plant notcomprising said introduced DNAs.

A plant, the genome of which comprises introduced DNAs encoding thefollowing enzymes:

a wild-type or deregulated aspartate kinase; and

a wild-type or deregulated threonine deaminase;

wherein each of said introduced DNAs is operatively linked to regulatorysignals that cause expression of said introduced DNAs; and

wherein cells of said plant produce an elevated amount of α-ketobutyratecompared to that in cells of a corresponding, wild-type plant notcomprising said introduced DNAs.

A plant, the genome of which comprises introduced DNAs encoding thefollowing enzymes:

a wild-type or deregulated aspartate kinase;

threonine synthase; and

a wild-type or deregulated threonine deaminase;

wherein each of said introduced DNAs is operatively linked to regulatorysignals that cause expression of said introduced DNAs; and

wherein cells of said plant produce an elevated amount of α-ketobutyratecompared to that in cells of a corresponding, wild-type plant notcomprising said introduced DNAs.

A plant, the genome of which comprises introduced DNAs encoding thefollowing enzymes:

a wild-type or deregulated aspartate kinase;

homoserine dehydrogenase; and

a wild-type or deregulated threonine deaminase;

wherein each of said introduced DNAs is operatively linked to regulatorysignals that cause expression of said introduced DNAs; and

wherein cells of said plant produce an increased amount ofα-keto-butyrate compared to that in cells of a corresponding, wild-typeplant not comprising said introduced DNAs.

A plant, the genome of which comprises introduced DNAs encoding thefollowing enzymes:

a wild-type or deregulated aspartate kinase;

homoserine dehydrogenase;

threonine synthase; and

a wild-type or deregulated threonine deaminase;

wherein each of said introduced DNAs is operatively linked to regulatorysignals that cause expression of said introduced DNAs; and

wherein cells of said plant produce an increased amount ofα-keto-butyrate compared to that in cells of a corresponding, wild-typeplant not comprising said introduced DNAs.

A plant, the genome of which comprises introduced DNAs encoding thefollowing enzymes:

a wild-type or deregulated threonine deaminase; and

an α-ketoacid decarboxylase E1 subunit enzyme that exhibits improvedbinding and decarboxylating activities with α-ketobutyrate compared withpyruvate decarboxylase E1 subunit naturally present in pyruvatedehydrogenase complex of said plant;

wherein each of said introduced DNAs is operatively linked to regulatorysignals that cause expression of said introduced DNAs; and

wherein cells of said plant produce an elevated amount of propionyl-CoAcompared to that in cells of a corresponding, wild-type plant notcomprising said introduced DNAs.

The foregoing plant, wherein said α-ketoacid decarboxylase E1 subunitthat exhibits improved binding and decarboxylating activities withα-ketobutyrate is a branched chain α-ketoacid decarboxylase E1 subunitsuch as that from bovine kidney, Pseudomonas putida, or Bacillussubtilis.

A plant, the genome of which comprises introduced DNAs encoding thefollowing enzymes:

a wild-type or deregulated threonine deaminase;

an α-ketoacid decarboxylase E1 subunit that exhibits improved bindingand decarboxylating activities with α-ketobutyrate compared withpyruvate decarboxylase E1 subunit naturally present in pyruvatedehydrogenase complex in said plant; and

a dihydrolipoyl transacylase E2 subunit exhibiting improved transacylaseactivity with α-ketobutyrate compared with dihydrolipoyl transacetylaseE2 subunit naturally present in pyruvate dehydrogenase complex in saidplant;

wherein each of said introduced DNAs is operatively linked to regulatorysignals that cause expression of said introduced DNAs; and

wherein cells of said plant produce an elevated amount of propionyl-CoAcompared to that in cells of a corresponding, wild-type plant notcomprising said introduced DNAs.

The foregoing plant, wherein said α-ketoacid decarboxylase E1 subunitthat exhibits improved binding and decarboxylating activities withα-ketobutyrate is a branched chain α-ketoacid decarboxylase E1 subunitsuch as that from bovine kidney, Pseudomonas putida, or Bacillussubtilis, and said dihydrolipoyl transacylase E2 subunit exhibitingimproved transacylase activity with α-ketobutyrate is a branched chaindihydrolipoyl transacylase E2 subunit such as that from bovine kidney,Pseudomonas putida, or Bacillus subtilis.

A plant, the genome of which comprises introduced DNAs encoding thefollowing enzymes:

a wild-type or deregulated threonine deaminase; and

pyruvate oxidase;

wherein each of said introduced DNAs is operatively linked to regulatorysignals that cause expression of said introduced DNAs; and

wherein cells of said plant produce an elevated amount of propionyl-CoAcompared that in cells of a corresponding, wild-type plant notcomprising said introduced DNAs.

A plant, the genome of which comprises introduced DNAs encoding thefollowing enzymes:

threonine synthase;

a wild-type or deregulated threonine deaminase; and

pyruvate oxidase;

wherein each of said introduced DNAs is operatively linked to regulatorysignals that cause expression of said introduced DNAs; and

wherein cells of said plant produce an elevated amount of propionyl-CoAcompared to that in cells of a corresponding, wild-type plant notcomprising said introduced DNAs.

A plant, the genome of which comprises introduced DNAs encoding thefollowing enzymes:

a wild-type or deregulated threonine deaminase;

pyruvate oxidase; and

an acyl-CoA synthetase;

wherein each of said introduced DNAs is operatively linked to regulatorysignals that cause expression of said introduced DNAs; and

wherein cells of said plant produce an elevated amount of propionyl-CoAcompared to that in cells of a corresponding, wild-type plant notcomprising said introduced DNAs.

A plant, the genome of which comprises introduced DNAs encoding thefollowing enzymes:

threonine synthase;

a wild-type or deregulated threonine deaminase;

pyruvate oxidase; and

an acyl-CoA synthetase;

wherein each of said introduced DNAs is operatively linked to regulatorysignals that cause expression of said introduced DNAs; and

wherein cells of said plant produce an elevated amount of propionyl-CoAcompared to that in cells of a corresponding, wild-type plant notcomprising said introduced DNAs.

A plant, the genome of which comprises introduced DNAs encoding thefollowing enzymes:

a wild-type or deregulated threonine deaminase;

pyruvate oxidase;

a β-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA and β-ketothiolase capable of condensingacetyl-CoA and propionyl-CoA to produce β-ketovaleryl-CoA; or

a β-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA, and capable of condensing acetyl-CoA andpropionyl-CoA to produce β-ketovaleryl-CoA;

a β-ketoacyl-CoA reductase capable of reducing acetoacetyl-CoA andβ-ketovaleryl-CoA to produce β-hydroxybutyryl-CoA andβ-hydroxy-valeryl-CoA, respectively; and

a polyhydroxyalkanoate synthase capable of incorporatingβ-hydroxybutyryl-CoA and β-hydroxyvaleryl-CoA into P(3HB-co-3HV)copolymer;

wherein each of said introduced DNAs is operatively linked to regulatorysignals that cause expression of said introduced DNAs; and

wherein said plant produces P(3HB-co-3HV) copolymer.

A plant, the genome of which comprises introduced DNAs encoding thefollowing enzymes:

threonine synthase;

a wild-type or deregulated threonine deaminase;

pyruvate oxidase;

a β-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA and a β-ketothiolase capable of condensingacetyl-CoA and propionyl-CoA to produce β-ketovaleryl-CoA; or

a β-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA, and capable of condensing acetyl-CoA andpropionyl-CoA to produce β-ketovaleryl-CoA;

a β-ketoacyl-CoA reductase capable of reducing acetoacetyl-CoA andβ-ketovaleryl-CoA to produce β-hydroxybutyryl-CoA andβ-hydroxy-valeryl-CoA, respectively; and

a polyhydroxyalkanoate synthase capable of incorporatingβ-hydroxybutyryl-CoA and β-hydroxyvaleryl-CoA into P(3HB-co-3HV)copolymer;

wherein each of said introduced DNAs is operatively linked to regulatorysignals that cause expression of said introduced DNAs; and

wherein said plant produces P(3HB-co-3HV) copolymer.

A plant, the genome of which comprises introduced DNAs encoding thefollowing enzymes:

a β-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA and a β-ketothiolase capable of condensingacetyl-CoA and propionyl-CoA to produce β-ketovaleryl-CoA; or

a β-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA, and capable of condensing acetyl-CoA andpropionyl-CoA to produce β-ketovaleryl-CoA;

a β-ketoacyl-CoA reductase capable of reducing acetoacetyl-CoA andβ-ketovaleryl-CoA to produce β-hydroxybutyryl-CoA andβ-hydroxy-valeryl-CoA, respectively; and

a polyhydroxyalkanoate synthase capable of incorporatingβ-hydroxybutyryl-CoA and β-hydroxyvaleryl-CoA into P(3HB-co-3HV)copolymer;

wherein each of said introduced DNAs is operatively linked to regulatorysignals that cause expression of said introduced DNAs; and

wherein said plant produces P(3HB-co-3HV) copolymer.

The foregoing plant, the genome of which further comprises introducedDNA encoding a wild-type or deregulated threonine deaminase;

wherein said introduced DNA is operatively linked to regulatory signalsthat cause expression of said introduced DNA; and

wherein said plant produces P(3HB-co-3HV) copolymer.

A plant, the genome of which comprises introduced DNAs encoding thefollowing enzymes:

threonine synthase;

a wild-type or deregulated threonine deaminase;

a β-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA and a β-ketothiolase capable of condensingacetyl-CoA and propionyl-CoA to produce β-ketovaleryl-CoA; or

a β-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA, and capable of condensing acetyl-CoA andpropionyl-CoA to produce β-ketovaleryl-CoA;

a β-ketoacyl-CoA reductase capable of reducing acetoacetyl-CoA andβ-ketovaleryl-CoA to produce β-hydroxybutyryl-CoA andβ-hydroxy-valeryl-CoA, respectively; and

a polyhydroxyalkanoate synthase capable of incorporatingβ-hydroxybutyryl-CoA and β-hydroxyvaleryl-CoA into P(3HB-co-3HV)copolymer;

wherein each of said introduced DNAs is operatively linked to regulatorysignals that cause expression of said introduced DNAs; and

wherein said plant produces P(3HB-co-3HV) copolymer.

Any of the foregoing plants, wherein the β-ketothiolase capable ofcondensing acetyl-CoA and propionyl-CoA to produce β-ketovaleryl-CoA isBktB.

Any of the foregoing plants, wherein said β-ketoacyl-CoA reductase isobtainable from a microorganism selected from the group consisting ofAlcaligenes eutrophus, Alcaligenes faecalis, Aphanothece sp.,Azotobacter vinelandii, Bacillus cereus, Bacillus megaterium,Beijerinkia indica, Derxia gummosa, Methylobacterium sp., Microcoleussp., Nocardia corallina, Pseudomonas cepacia, Pseudomonas extorquens,Pseudomonas oleovorans, Rhodobacter sphaeroides, Rhodobacter capsulatus,Rhodospirillum rubrum, and Thiocapsa pfennigii.

Any of the foregoing plants, wherein said polyhydroxyalkanoate synthaseis obtainable from a microorganism selected from the group consisting ofAlcaligenes eutrophus, Alcaligenes faecalis, Aphanothece sp.,Azotobacter vinelandii, Bacillus cereus, Bacillus megaterium,Beijerinkia indica, Derxia gummosa, Methylobacterium sp., Microcoleussp., Nocardia corallina, Pseudomonas cepacia, Pseudomonas extorquens,Pseudomonas oleovorans, Rhodobacter sphaeroides, Rhodobacter capsulatus,Rhodospirillum rubrum, and Thiocapsa pfennigii.

A plant, the genome of which comprises introduced DNA encoding awild-type or deregulated threonine deaminase enzyme;

wherein said introduced DNA is operatively linked to regulatory signalsthat cause expression of said introduced DNA; and

wherein cells of said plant produce an increased amount ofα-keto-butyrate compared to that in cells of a corresponding, wild-typeplant not comprising said introduced DNA.

A plant, the genome of which comprises introduced DNAs encoding thefollowing enzymes:

threonine synthase; and

a wild-type or deregulated threonine deaminase;

wherein said introduced DNAs are operatively linked to regulatorysignals that cause expression of said introduced DNAs; and

wherein cells of said plant produce an increased amount ofα-keto-butyrate compared to that in cells of a corresponding, wild-typeplant not comprising said introduced DNAs.

A plant, the genome of which comprises introduced DNAs encoding thefollowing enzymes:

a wild-type or deregulated aspartate kinase;

homoserine dehydrogenase;

a wild-type or deregulated threonine deaminase; and

pyruvate oxidase;

wherein each of said introduced DNAs is operatively linked to regulatorysignals that cause expression of said introduced DNAs; and

wherein cells of said plant produce an increased amount of propionyl-CoAcompared to that in cells of a corresponding, wild-type plant notcomprising said introduced DNAs.

A plant, the genome of which comprises introduced DNAs encoding thefollowing enzymes:

a wild-type or deregulated aspartate kinase;

homoserine dehydrogenase;

threonine synthase;

a wild-type or deregulated threonine deaminase; and

pyruvate oxidase;

wherein each of said introduced DNAs is operatively linked to regulatorysignals that cause expression of said introduced DNAs; and

wherein cells of said plant produce an increased amount of propionyl-CoAcompared to that in cells of a corresponding, wild-type plant notcomprising said introduced DNAs.

A plant, the genome of which comprises introduced DNAs encoding thefollowing enzymes:

a wild-type or deregulated aspartate kinase;

homoserine dehydrogenase;

a wild-type or deregulated threonine deaminase;

pyruvate oxidase; and

an acyl-CoA synthetase;

wherein each of said introduced DNAs is operatively linked to regulatorysignals that cause expression of said introduced DNAs; and

wherein cells of said plant produce an increased amount of propionyl-CoAcompared to that in cells of a corresponding, wild-type plant notcomprising said introduced DNAs.

A plant, the genome of which comprises introduced DNAs encoding thefollowing enzymes:

a wild-type or deregulated aspartate kinase;

homoserine dehydrogenase;

threonine synthase;

a wild-type or deregulated threonine deaminase;

pyruvate oxidase; and

an acyl-CoA synthetase;

wherein each of said introduced DNAs is operatively linked to regulatorysignals that cause expression of said introduced DNAs; and

wherein cells of said plant produce an increased amount of propionyl-CoAcompared to that in cells of a corresponding, wild-type plant notcomprising said introduced DNAs.

A plant, the genome of which comprises introduced DNAs encoding thefollowing enzymes:

a wild-type or deregulated threonine deaminase; and

an α-ketoacid decarboxylase E1 subunit that exhibits improved bindingand decarboxylating activities with α-ketobutyrate compared withpyruvate decarboxylase E1 subunit naturally present in pyruvatedehydrogenase complex in said plant;

wherein each of said introduced DNAs is operatively linked to regulatorysignals that cause expression of said introduced DNAs; and

wherein cells of said plant produce an increased amount of propionyl-CoA compared to that in cells of a corresponding, wild-type plant notcomprising said introduced DNAs.

A plant, the genome of which comprises introduced DNAs encoding thefollowing enzymes:

threonine synthase;

a wild-type or deregulated threonine deaminase; and

an α-ketoacid decarboxylase E1 subunit that exhibits improved bindingand decarboxylating activities with α-ketobutyrate compared withpyruvate decarboxylase E1 subunit naturally present in pyruvatedehydrogenase complex in said plant;

wherein each of said introduced DNAs is operatively linked to regulatorysignals that cause expression of said introduced DNAs; and

wherein cells of said plant produce an increased amount of propionyl-CoA compared to that in cells of a corresponding, wild-type plant notcomprising said introduced DNAs.

A plant, the genome of which comprises introduced DNAs encoding thefollowing enzymes:

a wild-type or deregulated aspartate kinase;

a wild-type or deregulated threonine deaminase; and

an α-ketoacid decarboxylase E1 subunit that exhibits improved bindingand decarboxylating activities with α-ketobutyrate compared withpyruvate decarboxylase E1 subunit naturally present in pyruvatedehydrogenase complex in said plant;

wherein each of said introduced DNAs is operatively linked to regulatorysignals that cause expression of said introduced DNAs; and

wherein cells of said plant produce an increased amount of propionyl-CoAcompared to that in cells of a corresponding, wild-type plant notcomprising said introduced DNAs.

A plant, the genome of which comprises introduced DNAs encoding thefollowing enzymes:

a wild-type or deregulated aspartate kinase;

threonine synthase;

a wild-type or deregulated threonine deaminase; and

an α-ketoacid decarboxylase E1 subunit that exhibits improved bindingand decarboxylating activities with α-ketobutyrate compared withpyruvate decarboxylase E1 subunit naturally present in pyruvatedehydrogenase complex in said plant;

wherein each of said introduced DNAs is operatively linked to regulatorysignals that cause expression of said introduced DNAs; and

wherein cells of said plant produce an increased amount of propionyl-CoAcompared to that in cells of a corresponding, wild-type plant notcomprising said introduced DNAs.

A plant, the genome of which comprises introduced DNAs encoding thefollowing enzymes:

a wild-type or deregulated aspartate kinase;

homoserine dehydrogenase;

a wild-type or deregulated threonine deaminase; and

an α-ketoacid decarboxylase E1 subunit that exhibits improved bindingand decarboxylating activities with α-ketobutyrate compared withpyruvate decarboxylase E1 subunit naturally present in pyruvatedehydrogenase complex in said plant;

wherein each of said introduced DNAs is operatively linked to regulatorysignals that cause expression of said introduced DNAs; and

wherein cells of said plant produce an increased amount of propionyl-CoAcompared to that in cells of a corresponding, wild-type plant notcomprising said introduced DNAs.

A plant, the genome of which comprises introduced DNAs encoding thefollowing enzymes:

a wild-type or deregulated aspartate kinase;

homoserine dehydrogenase;

threonine synthase;

a wild-type or deregulated threonine deaminase; and

an α-ketoacid decarboxylase E1 subunit that exhibits improved bindingand decarboxylating activities with α-ketobutyrate compared withpyruvate decarboxylase E1 subunit naturally present in pyruvatedehydrogenase complex in said plant;

wherein each of said introduced DNAs is operatively linked to regulatorysignals that cause expression of said introduced DNAs; and

wherein cells of said plant produce an increased amount of propionyl-CoAcompared to that in cells of a corresponding, wild-type plant notcomprising said introduced DNAs.

A plant, the genome of which comprises introduced DNAs encoding thefollowing enzymes:

a wild-type or deregulated threonine deaminase;

pyruvate oxidase; and

an α-ketoacid decarboxylase E1 subunit that exhibits improved bindingand decarboxylating activities with α-ketobutyrate compared withpyruvate decarboxylase E1 subunit naturally present in pyruvatedehydrogenase complex in said plant;

wherein each of said introduced DNAs is operatively linked to regulatorysignals that cause expression of said introduced DNAs; and

wherein cells of said plant produce an increased amount of propionyl-CoAcompared to that in cells of a corresponding, wild-type plant notcomprising said introduced DNAs.

A plant, the genome of which comprises introduced DNAs encoding thefollowing enzymes:

threonine synthase;

a wild-type or deregulated threonine deaminase;

pyruvate oxidase; and

an α-ketoacid decarboxylase E1 subunit that exhibits improved bindingand decarboxylating activities with α-ketobutyrate compared withpyruvate decarboxylase E1 subunit naturally present in pyruvatedehydrogenase complex in said plant;

wherein each of said introduced DNAs is operatively linked to regulatorysignals that cause expression of said introduced DNAs; and

wherein cells of said plant produce an increased amount of propionyl-CoAcompared to that in cells of a corresponding, wild-type plant notcomprising said introduced DNAs.

A plant, the genome of which comprises introduced DNAs encoding thefollowing enzymes:

a wild-type or deregulated threonine deaminase;

pyruvate oxidase;

an acyl-CoA synthetase; and

an α-ketoacid decarboxylase E1 subunit that exhibits improved bindingand decarboxylating activities with α-ketobutyrate compared withpyruvate decarboxylase E1 subunit naturally present in pyruvatedehydrogenase complex in said plant;

wherein each of said introduced DNAs is operatively linked to regulatorysignals that cause expression of said introduced DNAs; and

wherein cells of said plant produce an increased amount of propionyl-CoAcompared to that in cells of a corresponding, wild-type plant notcomprising said introduced DNAs.

A plant, the genome of which comprises introduced DNAs encoding thefollowing enzymes:

threonine synthase;

a wild-type or deregulated threonine deaminase;

pyruvate oxidase;

an acyl-CoA synthetase; and

an α-ketoacid decarboxylase E1 subunit that exhibits improved bindingand decarboxylating activities with α-ketobutyrate compared withpyruvate decarboxylase E1 subunit naturally present in pyruvatedehydrogenase complex in said plant;

wherein each of said introduced DNAs is operatively linked to regulatorysignals that cause expression of said introduced DNAs; and

wherein cells of said plant produce an increased amount of propionyl-CoAcompared to that in cells of a corresponding, wild-type plant notcomprising said introduced DNAs.

A plant, the genome of which comprises introduced DNAs encoding thefollowing enzymes:

a wild-type or deregulated aspartate kinase;

homoserine dehydrogenase;

a wild-type or deregulated threonine deaminase;

pyruvate oxidase; and

an α-ketoacid decarboxylase E1 subunit that exhibits improved bindingand decarboxylating activities with α-ketobutyrate compared withpyruvate decarboxylase E1 subunit naturally present in pyruvatedehydrogenase complex in said plant;

wherein each of said introduced DNAs is operatively linked to regulatorysignals that cause expression of said introduced DNAs; and

wherein cells of said plant produce an increased amount of propionyl-CoAcompared to that in cells of a corresponding, wild-type plant notcomprising said introduced DNAs.

A plant, the genome of which comprises introduced DNAs encoding thefollowing enzymes:

a wild-type or deregulated aspartate kinase;

homoserine dehydrogenase;

threonine synthase;

a wild-type or deregulated threonine deaminase;

pyruvate oxidase; and

an α-ketoacid decarboxylase E1 subunit that exhibits improved bindingand decarboxylating activities with α-ketobutyrate compared withpyruvate decarboxylase E1 subunit naturally present in pyruvatedehydrogenase complex in said plant;

wherein each of said introduced DNAs is operatively linked to regulatorysignals that cause expression of said introduced DNAs; and

wherein cells of said plant produce an increased amount of propionyl-CoAcompared to that in cells of a corresponding, wild-type plant notcomprising said introduced DNAs.

A plant, the genome of which comprises introduced DNAs encoding thefollowing enzymes:

a wild-type or deregulated threonine deaminase;

a wild-type or deregulated aspartate kinase;

homoserine dehydrogenase;

pyruvate oxidase;

an acyl-CoA synthetase; and

an α-ketoacid decarboxylase E1 subunit that exhibits improved bindingand decarboxylating activities with α-ketobutyrate compared withpyruvate decarboxylase E1 subunit naturally present in pyruvatedehydrogenase complex in said plant;

wherein each of said introduced DNAs is operatively linked to regulatorysignals that cause expression of said introduced DNAs; and

wherein cells of said plant produce an increased amount of propionyl-CoAcompared to that in cells of a corresponding, wild-type plant notcomprising said introduced DNAs.

A plant, the genome of which comprises introduced DNAs encoding thefollowing enzymes:

a wild-type or deregulated threonine deaminase;

a wild-type or deregulated aspartate kinase;

homoserine dehydrogenase;

threonine synthase;

pyruvate oxidase;

an acyl-CoA synthetase; and

an α-ketoacid decarboxylase E1 subunit that exhibits improved bindingand decarboxylating activities with α-ketobutyrate compared withpyruvate decarboxylase E1 subunit naturally present in pyruvatedehydrogenase complex in said plant;

wherein each of said introduced DNAs is operatively linked to regulatorysignals that cause expression of said introduced DNAs; and

wherein cells of said plant produce an increased amount of propionyl-CoAcompared to that in cells of a corresponding, wild-type plant notcomprising said introduced DNAs.

A plant, the genome of which comprises introduced DNAs encoding thefollowing enzymes:

a wild-type or deregulated aspartate kinase;

a wild-type or deregulated threonine deaminase;

a β-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA and a β-ketothiolase capable of condensingacetyl-CoA and propionyl-CoA to produce β-ketovaleryl-CoA; or

a β-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA, and capable of condensing acetyl-CoA andpropionyl-CoA to produce β-ketovaleryl-CoA;

a β-ketoacyl-CoA reductase capable of reducing acetoacetyl-CoA andβ-ketovaleryl-CoA to produce β-hydroxybutyryl-CoA andβ-hydroxy-valeryl-CoA, respectively; and

a polyhydroxyalkanoate synthase capable of incorporatingβ-hydroxybutyryl-CoA and β-hydroxyvaleryl-CoA into P(3HB-co-3HV)copolymer;

wherein each of said introduced DNAs is operatively linked to regulatorysignals that cause expression of said introduced DNAs; and

wherein cells of said plant produce P(3HB-co-3HV) copolymer.

A plant, the genome of which comprises introduced DNAs encoding thefollowing enzymes:

a wild-type or deregulated aspartate kinase;

threonine synthase;

a wild-type or deregulated threonine deaminase;

a β-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA and a β-ketothiolase capable of condensingacetyl-CoA and propionyl-CoA to produce β-ketovaleryl-CoA; or

a β-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA, and capable of condensing acetyl-CoA andpropionyl-CoA to produce β-ketovaleryl-CoA;

a β-ketoacyl-CoA reductase capable of reducing acetoacetyl-CoA andβ-ketovaleryl-CoA to produce β-hydroxybutyryl-CoA andβ-hydroxy-valeryl-CoA, respectively; and

a polyhydroxyalkanoate synthase capable of incorporatingβ-hydroxybutyryl-CoA and β-hydroxyvaleryl-CoA into P(3HB-co-3HV)copolymer;

wherein each of said introduced DNAs is operatively linked to regulatorysignals that cause expression of said introduced DNAs; and

wherein cells of said plant produce P(3HB-co-3HV) copolymer.

A plant, the genome of which comprises introduced DNAs encoding thefollowing enzymes:

a wild-type or deregulated aspartate kinase;

homoserine dehydrogenase;

a wild-type or deregulated threonine deaminase;

a β-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA and a β-ketothiolase capable of condensingacetyl-CoA and propionyl-CoA to produce β-ketovaleryl-CoA; or

a β-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA, and capable of condensing acetyl-CoA andpropionyl-CoA to produce β-ketovaleryl-CoA;

a β-ketoacyl-CoA reductase capable of reducing acetoacetyl-CoA andβ-ketovaleryl-CoA to produce β-hydroxybutyryl-CoA andβ-hydroxy-valeryl-CoA, respectively; and

a polyhydroxyalkanoate synthase capable of incorporatingβ-hydroxybutyryl-CoA and β-hydroxyvaleryl-CoA into P(3HB-co-3HV)copolymer;

wherein each of said introduced DNAs is operatively linked to regulatorysignals that cause expression of said introduced DNAs; and

wherein cells of said plant produce P(3HB-co-3HV) copolymer.

A plant, the genome of which comprises introduced DNAs encoding thefollowing enzymes:

a wild-type or deregulated aspartate kinase;

homoserine dehydrogenase;

threonine synthase;

a wild-type or deregulated threonine deaminase;

a β-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA and a β-ketothiolase capable of condensingacetyl-CoA and propionyl-CoA to produce β-ketovaleryl-CoA; or

a β-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA, and capable of condensing acetyl-CoA andpropionyl-CoA to produce β-ketovaleryl-CoA;

a β-ketoacyl-CoA reductase capable of reducing acetoacetyl-CoA andβ-ketovaleryl-CoA to produce β-hydroxybutyryl-CoA andβ-hydroxy-valeryl-CoA, respectively; and

a polyhydroxyalkanoate synthase capable of incorporatingβ-hydroxybutyryl-CoA and β-hydroxyvaleryl-CoA into P(3HB-co-3HV)copolymer;

wherein each of said introduced DNAs is operatively linked to regulatorysignals that cause expression of said introduced DNAs; and

wherein cells of said plant produce P(3HB-co-3HV) copolymer.

A plant, the genome of which comprises introduced DNAs encoding thefollowing enzymes:

a wild-type or deregulated threonine deaminase;

pyruvate oxidase;

a β-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA and a β-ketothiolase capable of condensingacetyl-CoA and propionyl-CoA to produce β-ketovaleryl-CoA; or

a β-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA, and capable of condensing acetyl-CoA andpropionyl-CoA to produce β-ketovaleryl-CoA;

a β-ketoacyl-CoA reductase capable of reducing acetoacetyl-CoA andβ-ketovaleryl-CoA to produce β-hydroxybutyryl-CoA andβ-hydroxy-valeryl-CoA, respectively; and

a polyhydroxyalkanoate synthase capable of incorporatingβ-hydroxybutyryl-CoA and β-hydroxyvaleryl-CoA into P(3HB-co-3HV)copolymer;

wherein each of said introduced DNAs is operatively linked to regulatorysignals that cause expression of said introduced DNAs; and

wherein cells of said plant produce P(3HB-co-3HV) copolymer.

A plant, the genome of which comprises introduced DNAs encoding thefollowing enzymes:

threonine synthase;

a wild-type or deregulated threonine deaminase;

pyruvate oxidase;

a β-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA and a β-ketothiolase capable of condensingacetyl-CoA and propionyl-CoA to produce β-ketovaleryl-CoA; or

a β-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA, and capable of condensing acetyl-CoA andpropionyl-CoA to produce β-ketovaleryl-CoA;

a β-ketoacyl-CoA reductase capable of reducing acetoacetyl-CoA andβ-ketovaleryl-CoA to produce β-hydroxybutyryl-CoA andβ-hydroxyvaleryl-CoA, respectively; and

a polyhydroxyalkanoate synthase capable of incorporatingβ-hydroxybutyryl-CoA and β-hydroxyvaleryl-CoA into P(3HB-co-3HV)copolymer;

wherein each of said introduced DNAs is operatively linked to regulatorysignals that cause expression of said introduced DNAs; and

wherein cells of said plant produce P(3HB-co-3HV) copolymer.

A plant, the genome of which comprises introduced DNAs encoding thefollowing enzymes:

a wild-type or deregulated threonine deaminase;

pyruvate oxidase;

an acyl-CoA synthetase;

a β-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA and a β-ketothiolase capable of condensingacetyl-CoA and propionyl-CoA to produce β-ketovaleryl-CoA; or

a β-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA, and capable of condensing acetyl-CoA andpropionyl-CoA to produce β-ketovaleryl-CoA;

a β-ketoacyl-CoA reductase capable of reducing acetoacetyl-CoA andβ-ketovaleryl-CoA to produce β-hydroxybutyryl-CoA andβ-hydroxy-valeryl-CoA, respectively; and

a polyhydroxyalkanoate synthase capable of incorporatingβ-hydroxybutyryl-CoA and β-hydroxyvaleryl-CoA into P(3HB-co-3HV)copolymer;

wherein each of said introduced DNAs is operatively linked to regulatorysignals that cause expression of said introduced DNAs; and

wherein cells of said plant produce P(3HB-co-3HV) copolymer.

A plant, the genome of which comprises introduced DNAs encoding thefollowing enzymes:

threonine synthase;

a wild-type or deregulated threonine deaminase;

pyruvate oxidase;

an acyl-CoA synthetase;

a β-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA and a β-ketothiolase capable of condensingacetyl-CoA and propionyl-CoA to produce β-ketovaleryl-CoA; or

a β-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA, and capable of condensing acetyl-CoA andpropionyl-CoA to produce β-ketovaleryl-CoA;

a β-ketoacyl-CoA reductase capable of reducing acetoacetyl-CoA andβ-ketovaleryl-CoA to produce β-hydroxybutyryl-CoA andβ-hydroxy-valeryl-CoA, respectively; and

a polyhydroxyalkanoate synthase capable of incorporatingβ-hydroxybutyryl-CoA and β-hydroxyvaleryl-CoA into P(3HB-co-3HV)copolymer;

wherein each of said introduced DNAs is operatively linked to regulatorysignals that cause expression of said introduced DNAs; and

wherein cells of said plant produce P(3HB-co-3HV) copolymer.

A plant, the genome of which comprises introduced DNAs encoding thefollowing enzymes:

a wild-type or deregulated aspartate kinase;

homoserine dehydrogenase;

a wild-type or deregulated threonine deaminase;

pyruvate oxidase;

a β-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA and a β-ketothiolase capable of condensingacetyl-CoA and propionyl-CoA to produce β-ketovaleryl-CoA; or

a β-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA, and capable of condensing acetyl-CoA andpropionyl-CoA to produce β-ketovaleryl-CoA;

a β-ketoacyl-CoA reductase capable of reducing acetoacetyl-CoA andβ-ketovaleryl-CoA to produce β-hydroxybutyryl-CoA andβ-hydroxy-valeryl-CoA, respectively; and

a polyhydroxyalkanoate synthase capable of incorporatingβ-hydroxybutyryl-CoA and β-hydroxyvaleryl-CoA into P(3HB-co-3HV)copolymer;

wherein each of said introduced DNAs is operatively linked to regulatorysignals that cause expression of said introduced DNAs; and

wherein cells of said plant produce P(3HB-co-3HV) copolymer.

A plant, the genome of which comprises introduced DNAs encoding thefollowing enzymes:

a wild-type or deregulated aspartate kinase;

homoserine dehydrogenase;

threonine synthase;

a wild-type or deregulated threonine deaminase;

pyruvate oxidase;

a β-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA and a β-ketothiolase capable of condensingacetyl-CoA and propionyl-CoA to produce β-ketovaleryl-CoA; or

a β-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA, and capable of condensing acetyl-CoA andpropionyl-CoA to produce β-ketovaleryl-CoA;

a β-ketoacyl-CoA reductase capable of reducing acetoacetyl-CoA andβ-ketovaleryl-CoA to produce β-hydroxybutyryl-CoA andβ-hydroxy-valeryl-CoA, respectively; and

a polyhydroxyalkanoate synthase capable of incorporatingβ-hydroxybutyryl-CoA and β-hydroxyvaleryl-CoA into P(3HB-co-3HV)copolymer;

wherein each of said introduced DNAs is operatively linked to regulatorysignals that cause expression of said introduced DNAs; and

wherein cells of said plant produce P(3HB-co-3HV) copolymer.

A plant, the genome of which comprises introduced DNAs encoding thefollowing enzymes:

a wild-type or deregulated aspartate kinase;

homoserine dehydrogenase;

a wild-type or deregulated threonine deaminase;

pyruvate oxidase;

an acyl-CoA synthetase;

a β-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA and a β-ketothiolase capable of condensingacetyl-CoA and propionyl-CoA to produce β-ketovaleryl-CoA; or

a β-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA, and capable of condensing acetyl-CoA andpropionyl-CoA to produce β-ketovaleryl-CoA;

a β-ketoacyl-CoA reductase capable of reducing acetoacetyl-CoA andβ-ketovaleryl-CoA to produce β-hydroxybutyryl-CoA andβ-hydroxy-valeryl-CoA, respectively; and

a polyhydroxyalkanoate synthase capable of incorporatingβ-hydroxybutyryl-CoA and β-hydroxyvaleryl-CoA into P(3HB-co-3HV)copolymer;

wherein each of said introduced DNAs is operatively linked to regulatorysignals that cause expression of said introduced DNAs; and

wherein cells of said plant produce P(3HB-co-3HV) copolymer.

A plant, the genome of which comprises introduced DNAs encoding thefollowing enzymes:

a wild-type or deregulated aspartate kinase;

homoserine dehydrogenase;

threonine synthase;

a wild-type or deregulated threonine deaminase;

pyruvate oxidase;

an acyl-CoA synthetase;

a β-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA and a β-ketothiolase capable of condensingacetyl-CoA and propionyl-CoA to produce β-ketovaleryl-CoA; or

a β-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA, and capable of condensing acetyl-CoA andpropionyl-CoA to produce β-ketovaleryl-CoA;

a β-ketoacyl-CoA reductase capable of reducing acetoacetyl-CoA andβ-ketovaleryl-CoA to produce β-hydroxybutyryl-CoA andβ-hydroxy-valeryl-CoA, respectively; and

a polyhydroxyalkanoate synthase capable of incorporatingβ-hydroxybutyryl-CoA and β-hydroxyvaleryl-CoA into P(3HB-co-3HV)copolymer;

wherein each of said introduced DNAs is operatively linked to regulatorysignals that cause expression of said introduced DNAs; and

wherein cells of said plant produce P(3HB-co-3HV) copolymer.

A plant, the genome of which comprises introduced DNAs encoding thefollowing enzymes:

a wild-type or deregulated threonine deaminase;

an α-ketoacid decarboxylase E1 subunit that exhibits improved bindingand decarboxylating activities with α-ketobutyrate compared withpyruvate decarboxylase E1 subunit naturally present in pyruvatedehydrogenase complex in said plant;

a β-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA and a β-ketothiolase capable of condensingacetyl-CoA and propionyl-CoA to produce β-ketovaleryl-CoA; or

a β-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA, and capable of condensing acetyl-CoA andpropionyl-CoA to produce β-ketovaleryl-CoA;

a β-ketoacyl-CoA reductase capable of reducing acetoacetyl-CoA andβ-ketovaleryl-CoA to produce β-hydroxybutyryl-CoA andβ-hydroxy-valeryl-CoA, respectively; and

a polyhydroxyalkanoate synthase capable of incorporatingβ-hydroxybutyryl-CoA and β-hydroxyvaleryl-CoA into P(3HB-co-3HV)copolymer;

wherein each of said introduced DNAs is operatively linked to regulatorysignals that cause expression of said introduced DNAs; and

wherein cells of said plant produce P(3HB-co-3HV) copolymer.

A plant, the genome of which comprises introduced DNAs encoding thefollowing enzymes:

threonine synthase;

a wild-type or deregulated threonine deaminase;

an α-ketoacid decarboxylase E1 subunit that exhibits improved bindingand decarboxylating activities with α-ketobutyrate compared withpyruvate decarboxylase E1 subunit naturally present in pyruvatedehydrogenase complex in said plant;

a β-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA and a β-ketothiolase capable of condensingacetyl-CoA and propionyl-CoA to produce β-ketovaleryl-CoA; or

a β-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA, and capable of condensing acetyl-CoA andpropionyl-CoA to produce β-ketovaleryl-CoA;

a β-ketoacyl-CoA reductase capable of reducing acetoacetyl-CoA andβ-ketovaleryl-CoA to produce β-hydroxybutyryl-CoA andβ-hydroxy-valeryl-CoA, respectively; and

a polyhydroxyalkanoate synthase capable of incorporatingβ-hydroxybutyryl-CoA and β-hydroxyvaleryl-CoA into P(3HB-co-3HV)copolymer;

wherein each of said introduced DNAs is operatively linked to regulatorysignals that cause expression of said introduced DNAs; and

wherein cells of said plant produce P(3HB-co-3HV) copolymer.

A plant, the genome of which comprises introduced DNAs encoding thefollowing enzymes:

a wild-type or deregulated aspartate kinase;

a wild-type or deregulated threonine deaminase;

an α-ketoacid decarboxylase E1 subunit that exhibits improved bindingand decarboxylating activities with α-ketobutyrate compared withpyruvate decarboxylase E1 subunit naturally present in pyruvatedehydrogenase complex in said plant;

a β-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA and a β-ketothiolase capable of condensingacetyl-CoA and propionyl-CoA to produce β-ketovaleryl-CoA; or

a β-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA, and capable of condensing acetyl-CoA andpropionyl-CoA to produce β-ketovaleryl-CoA;

a β-ketoacyl-CoA reductase capable of reducing acetoacetyl-CoA andβ-ketovaleryl-CoA to produce β-hydroxybutyryl-CoA andβ-hydroxy-valeryl-CoA, respectively; and

a polyhydroxyalkanoate synthase capable of incorporatingβ-hydroxybutyryl-CoA and β-hydroxyvaleryl-CoA into P(3HB-co-3HV)copolymer;

wherein each of said introduced DNAs is operatively linked to regulatorysignals that cause expression of said introduced DNAs; and

wherein cells of said plant produce P(3HB-co-3HV) copolymer.

A plant, the genome of which comprises introduced DNAs encoding thefollowing enzymes:

a wild-type or deregulated aspartate kinase;

threonine synthase;

a wild-type or deregulated threonine deaminase;

an α-ketoacid decarboxylase E1 subunit that exhibits improved bindingand decarboxylating activities with α-ketobutyrate compared withpyruvate decarboxylase E1 subunit naturally present in pyruvatedehydrogenase complex in said plant;

a β-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA and a β-ketothiolase capable of condensingacetyl-CoA and propionyl-CoA to produce β-ketovaleryl-CoA; or

a β-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA, and capable of condensing acetyl-CoA andpropionyl-CoA to produce β-ketovaleryl-CoA;

a β-ketoacyl-CoA reductase capable of reducing acetoacetyl-CoA andβ-ketovaleryl-CoA to produce β-hydroxybutyryl-CoA andβ-hydroxy-valeryl-CoA, respectively; and

a polyhydroxyalkanoate synthase capable of incorporatingβ-hydroxybutyryl-CoA and β-hydroxyvaleryl-CoA into P(3HB-co-3HV)copolymer;

wherein each of said introduced DNAs is operatively linked to regulatorysignals that cause expression of said introduced DNAs; and

wherein cells of said plant produce P(3HB-co-3HV) copolymer.

A plant, the genome of which comprises introduced DNAs encoding thefollowing enzymes:

a wild-type or deregulated aspartate kinase;

homoserine dehydrogenase;

a wild-type or deregulated threonine deaminase;

an α-ketoacid decarboxylase E1 subunit that exhibits improved bindingand decarboxylating activities with α-ketobutyrate compared withpyruvate decarboxylase E1 subunit naturally present in pyruvatedehydrogenase complex in said plant;

a β-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA and a β-ketothiolase capable of condensingacetyl-CoA and propionyl-CoA to produce β-ketovaleryl-CoA; or

a β-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA, and capable of condensing acetyl-CoA andpropionyl-CoA to produce β-ketovaleryl-CoA;

a β-ketoacyl-CoA reductase capable of reducing acetoacetyl-CoA andβ-ketovaleryl-CoA to produce β-hydroxybutyryl-CoA andβ-hydroxy-valeryl-CoA, respectively; and

a polyhydroxyalkanoate synthase capable of incorporatingβ-hydroxybutyryl-CoA and β-hydroxyvaleryl-CoA into P(3HB-co-3HV)copolymer;

wherein each said introduced DNAs is operatively linked to regulatorysignals that cause expression of said introduced DNAs; and

wherein cells of said plant produce P(3HB-co-3HV) copolymer.

A plant, the genome of which comprises introduced DNAs encoding thefollowing enzymes:

a wild-type or deregulated aspartate kinase;

homoserine dehydrogenase;

threonine synthase;

a wild-type or deregulated threonine deaminase;

an α-ketoacid decarboxylase E1 subunit that exhibits improved bindingand decarboxylating activities with α-ketobutyrate compared withpyruvate decarboxylase E1 subunit naturally present in pyruvatedehydrogenase complex in said plant;

a β-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA and a β-ketothiolase capable of condensingacetyl-CoA and propionyl-CoA to produce β-ketovaleryl-CoA; or

a β-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA, and capable of condensing acetyl-CoA andpropionyl-CoA to produce β-ketovaleryl-CoA;

a β-ketoacyl-CoA reductase capable of reducing acetoacetyl-CoA andβ-ketovaleryl-CoA to produce β-hydroxybutyryl-CoA andβ-hydroxy-valeryl-CoA, respectively; and

a polyhydroxyalkanoate synthase capable of incorporatingβ-hydroxybutyryl-CoA and β-hydroxyvaleryl-CoA into P(3HB-co-3HV)copolymer;

wherein each said introduced DNAs is operatively linked to regulatorysignals that cause expression of said introduced DNAs; and

wherein cells of said plant produce P(3HB-co-3HV) copolymer.

A plant, the genome of which comprises introduced DNAs encoding thefollowing enzymes:

a wild-type or deregulated threonine deaminase;

pyruvate oxidase;

an α-ketoacid decarboxylase E1 subunit that exhibits improved bindingand decarboxylating activities with α-ketobutyrate compared withpyruvate decarboxylase E1 subunit naturally present in pyruvatedehydrogenase complex in said plant;

a β-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA and a β-ketothiolase capable of condensingacetyl-CoA and propionyl-CoA to produce β-ketovaleryl-CoA; or

a β-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA, and capable of condensing acetyl-CoA andpropionyl-CoA to produce β-ketovaleryl-CoA;

a β-ketoacyl-CoA reductase capable of reducing acetoacetyl-CoA andβ-ketovaleryl-CoA to produce β-hydroxybutyryl-CoA andβ-hydroxy-valeryl-CoA, respectively; and

a polyhydroxyalkanoate synthase capable of incorporatingβ-hydroxybutyryl-CoA and β-hydroxyvaleryl-CoA into P(3HB-co-3HV)copolymer;

wherein each of said introduced DNAs is operatively linked to regulatorysignals that cause expression of said introduced DNAs; and

wherein cells of said plant produce P(3HB-co-3HV) copolymer.

A plant, the genome of which comprises introduced DNAs encoding thefollowing enzymes:

threonine synthase;

a wild-type or deregulated threonine deaminase;

pyruvate oxidase;

an α-ketoacid decarboxylase E1 subunit that exhibits improved bindingand decarboxylating activities with α-ketobutyrate compared withpyruvate decarboxylase E1 subunit naturally present in pyruvatedehydrogenase complex in said plant;

a β-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA and a β-ketothiolase capable of condensingacetyl-CoA and propionyl-CoA to produce β-ketovaleryl-CoA; or

a β-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA, and capable of condensing acetyl-CoA andpropionyl-CoA to produce β-ketovaleryl-CoA;

a β-ketoacyl-CoA reductase capable of reducing acetoacetyl-CoA andβ-ketovaleryl-CoA to produce β-hydroxybutyryl-CoA andβ-hydroxy-valeryl-CoA, respectively; and

a polyhydroxyalkanoate synthase capable of incorporatingβ-hydroxybutyryl-CoA and β-hydroxyvaleryl-CoA into P(3HB-co-3HV)copolymer;

wherein each of said introduced DNAs is operatively linked to regulatorysignals that cause expression of said introduced DNAs; and

wherein cells of said plant produce P(3HB-co-3HV) copolymer.

A plant, the genome of which comprises introduced DNAs encoding thefollowing enzymes:

a wild-type or deregulated threonine deaminase;

pyruvate oxidase;

an acyl-CoA synthetase;

an α-ketoacid decarboxylase E1 subunit that exhibits improved bindingand decarboxylating activities with α-ketobutyrate compared withpyruvate decarboxylase E1 subunit naturally present in pyruvatedehydrogenase complex in said plant;

a β-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA and a β-ketothiolase capable of condensingacetyl-CoA and propionyl-CoA to produce β-ketovaleryl-CoA; or

a β-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA, and capable of condensing acetyl-CoA andpropionyl-CoA to produce β-ketovaleryl-CoA;

a β-ketoacyl-CoA reductase capable of reducing acetoacetyl-CoA andβ-ketovaleryl-CoA to produce β-hydroxybutyryl-CoA andβ-hydroxy-valeryl-CoA, respectively; and

a polyhydroxyalkanoate synthase capable of incorporatingβ-hydroxybutyryl-CoA and β-hydroxyvaleryl-CoA into P(3HB-co-3HV)copolymer;

wherein each of said introduced DNAs is operatively linked to regulatorysignals that cause expression of said introduced DNAs; and

wherein cells of said plant produce P(3HB-co-3HV) copolymer.

A plant, the genome of which comprises introduced DNAs encoding thefollowing enzymes:

threonine synthase;

a wild-type or deregulated threonine deaminase;

pyruvate oxidase;

an acyl-CoA synthetase;

an α-ketoacid decarboxylase E1 subunit that exhibits improved bindingand decarboxylating activities with α-ketobutyrate compared withpyruvate decarboxylase E1 subunit naturally present in pyruvatedehydrogenase complex in said plant;

a β-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA and a β-ketothiolase capable of condensingacetyl-CoA and propionyl-CoA to produce β-ketovaleryl-CoA; or

a β-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA, and capable of condensing acetyl-CoA andpropionyl-CoA to produce β-ketovaleryl-CoA;

a β-ketoacyl-CoA reductase capable of reducing acetoacetyl-CoA andβ-ketovaleryl-CoA to produce β-hydroxybutyryl-CoA andβ-hydroxy-valeryl-CoA, respectively; and

a polyhydroxyalkanoate synthase capable of incorporatingβ-hydroxybutyryl-CoA and β-hydroxyvaleryl-CoA into P(3HB-co-3HV)copolymer;

wherein each of said introduced DNAs is operatively linked to regulatorysignals that cause expression of said introduced DNAs; and

wherein cells of said plant produce P(3HB-co-3HV) copolymer.

A plant, the genome of which comprises introduced DNAs encoding thefollowing enzymes:

a wild-type or deregulated aspartate kinase;

homoserine dehydrogenase;

a wild-type or deregulated threonine deaminase;

pyruvate oxidase;

an α-ketoacid decarboxylase E1 subunit that exhibits improved bindingand decarboxylating activities with α-ketobutyrate compared withpyruvate decarboxylase E1 subunit naturally present in pyruvatedehydrogenase complex in said plant;

a β-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA and a β-ketothiolase capable of condensingacetyl-CoA and propionyl-CoA to produce β-ketovaleryl-CoA; or

a β-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA, and capable of condensing acetyl-CoA andpropionyl-CoA to produce β-ketovaleryl-CoA;

a β-ketoacyl-CoA reductase capable of reducing acetoacetyl-CoA andβ-ketovaleryl-CoA to produce β-hydroxybutyryl-CoA andβ-hydroxy-valeryl-CoA, respectively; and

a polyhydroxyalkanoate synthase capable of incorporatingβ-hydroxybutyryl-CoA and β-hydroxyvaleryl-CoA into P(3HB-co-3HV)copolymer;

wherein each of said introduced DNAs is operatively linked to regulatorysignals that cause expression of said introduced DNAs; and

wherein cells of said plant produce P(3HB-co-3HV) copolymer.

A plant, the genome of which comprises introduced DNAs encoding thefollowing enzymes:

a wild-type or deregulated aspartate kinase;

homoserine dehydrogenase;

threonine synthase;

a wild-type or deregulated threonine deaminase;

pyruvate oxidase;

an α-ketoacid decarboxylase E1 subunit that exhibits improved bindingand decarboxylating activities with α-ketobutyrate compared withpyruvate decarboxylase E1 subunit naturally present in pyruvatedehydrogenase complex in said plant;

a β-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA and a β-ketothiolase capable of condensingacetyl-CoA and propionyl-CoA to produce β-ketovaleryl-CoA; or

a β-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA, and capable of condensing acetyl-CoA andpropionyl-CoA to produce β-ketovaleryl-CoA;

a β-ketoacyl-CoA reductase capable of reducing acetoacetyl-CoA andβ-ketovaleryl-CoA to produce β-hydroxybutyryl-CoA andβ-hydroxy-valeryl-CoA, respectively; and

a polyhydroxyalkanoate synthase capable of incorporatingβ-hydroxybutyryl-CoA and β-hydroxyvaleryl-CoA into P(3HB-co-3HV)copolymer;

wherein each of said introduced DNAs is operatively linked to regulatorysignals that cause expression of said introduced DNAs; and

wherein cells of said plant produce P(3HB-co-3HV) copolymer.

A plant, the genome of which comprises introduced DNAs encoding thefollowing enzymes:

a wild-type or deregulated aspartate kinase;

homoserine dehydrogenase;

a wild-type or deregulated threonine deaminase;

pyruvate oxidase;

an acyl-CoA synthetase;

an α-ketoacid decarboxylase E1 subunit that exhibits improved bindingand decarboxylating activities with α-ketobutyrate compared withpyruvate decarboxylase E1 subunit naturally present in pyruvatedehydrogenase complex in said plant;

a β-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA and a β-ketothiolase capable of condensingacetyl-CoA and propionyl-CoA to produce β-ketovaleryl-CoA; or

a β-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA, and capable of condensing acetyl-CoA andpropionyl-CoA to produce β-ketovaleryl-CoA;

a β-ketoacyl-CoA reductase capable of reducing acetoacetyl-CoA andβ-ketovaleryl-CoA to produce β-hydroxybutyryl-CoA andβ-hydroxy-valeryl-CoA, respectively; and

a polyhydroxyalkanoate synthase capable of incorporatingβ-hydroxybutyryl-CoA and β-hydroxyvaleryl-CoA into P(3HB-co-3HV)copolymer;

wherein each of said introduced DNAs is operatively linked to regulatorysignals that cause expression of said introduced DNAs; and

wherein cells of said plant produce P(3HB-co-3HV) copolymer.

A plant, the genome of which comprises introduced DNAs encoding thefollowing enzymes:

a wild-type or deregulated aspartate kinase;

homoserine dehydrogenase;

threonine synthase;

a wild-type or deregulated threonine deaminase;

pyruvate oxidase;

an acyl-CoA synthetase;

an α-ketoacid decarboxylase E1 subunit that exhibits improved bindingand decarboxylating activities with α-ketobutyrate compared withpyruvate decarboxylase E1 subunit naturally present in pyruvatedehydrogenase complex in said plant;

a β-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA and a β-ketothiolase capable of condensingacetyl-CoA and propionyl-CoA to produce β-ketovaleryl-CoA; or

a β-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA, and capable of condensing acetyl-CoA andpropionyl-CoA to produce β-ketovaleryl-CoA;

a β-ketoacyl-CoA reductase capable of reducing acetoacetyl-CoA andβ-ketovaleryl-CoA to produce β-hydroxybutyryl-CoA andβ-hydroxy-valeryl-CoA, respectively; and

a polyhydroxyalkanoate synthase capable of incorporatingβ-hydroxybutyryl-CoA and β-hydroxyvaleryl-CoA into P(3HB-co-3HV)copolymer;

wherein each of said introduced DNAs is operatively linked to regulatorysignals that cause expression of said introduced DNAs; and

wherein cells of said plant produce P(3HB-co-3HV) copolymer.

Any of the foregoing plants, wherein each of said introduced DNAs isfurther operatively linked to a plastid transit peptide coding regioncapable of directing transport of said enzyme encoded thereby into aplastid.

A method of producing P(3HB-co-3HV)copolymer, comprising growing any ofthe foregoing plants, the genome of which comprises introduced DNAsencoding a β-ketothiolase, a β-ketoacyl-CoA reductase, and apolyhydroxyalkanoate synthase, and recovering said P(3HB-co-3HV)copolymer produced thereby.

The foregoing method, wherein said P(3HB-co-3HV)copolymer is recoveredfrom leaves or seeds of said plant.

A plant cell containing P(3HB-co-3HV) copolymer.

A plant comprising cells containing P(3HB-co-3HV) copolymer.

Seeds of the foregoing plant.

The foregoing plant, wherein said cells are located in leaves or seedsof said plant.

A plant, the genome of which comprises introduced DNAs encoding theenzymes PhbA, PhbB, and PhbC;

wherein each of said introduced DNAs is operatively linked to a plastidtransit peptide coding region capable of directing transport of saidenzymes into a plastid, and regulatory signals that cause expression ofsaid introduced DNAs in seeds of said plant; and

wherein P(3HB) homopolymer is produced in seeds of said plant.

A method of producing P(3HB) homopolymer, comprising growing theforegoing plant, and recovering said P(3HB) homopolymer producedthereby.

The foregoing method, wherein said P(3HB) homopolymer is recovered fromseeds of said plant.

A bacterium, the genome of which comprises introduced DNA encoding awild-type or deregulated aspartate kinase;

wherein said introduced DNA is operatively linked to regulatory signalsthat cause expression of said introduced DNA; and

wherein said bacterium produces an elevated amount of threonine comparedto that in a corresponding wild-type bacterium not comprising saidintroduced DNA.

A bacterium, the genome of which comprises introduced DNAs encoding thefollowing enzymes:

a wild-type or deregulated aspartate kinase; and

threonine synthase;

wherein said introduced DNAs are operatively linked to regulatorysignals that cause expression of said introduced DNAs; and

wherein said bacterium produces an elevated amount of threonine comparedto that in a corresponding wild-type bacterium not comprising saidintroduced DNAs.

A bacterium, the genome of which comprises introduced DNAs encoding thefollowing enzymes:

a wild-type or deregulated aspartate kinase; and

homoserine dehydrogenase;

wherein said introduced DNAs are operatively linked to regulatorysignals that cause expression of said introduced DNAs; and

wherein said bacterium produces an elevated amount of threonine comparedto that in a corresponding wild-type bacterium not comprising saidintroduced DNAs.

A bacterium, the genome of which comprises introduced DNAs encoding thefollowing enzymes:

a wild-type or deregulated aspartate kinase;

homoserine dehydrogenase; and

threonine synthase;

wherein said introduced DNAs are operatively linked to regulatorysignals that cause expression of said introduced DNAs; and

wherein said bacterium produces an elevated amount of threonine comparedto that in a corresponding wild-type bacterium not comprising saidintroduced DNAs.

A bacterium, the genome of which comprises introduced DNAs encoding thefollowing enzymes:

a wild-type or deregulated aspartate kinase; and

a wild-type or deregulated threonine deaminase;

wherein said introduced DNAs are operatively linked to regulatorysignals that cause expression of said introduced DNAs; and

wherein said bacterium produces an elevated amount of α-keto-butyratecompared to that in a corresponding wild-type bacterium not comprisingsaid introduced DNAs.

A bacterium, the genome of which comprises introduced DNAs encoding thefollowing enzymes:

a wild-type or deregulated aspartate kinase;

threonine synthase; and

a wild-type or deregulated threonine deaminase;

wherein said introduced DNAs are operatively linked to regulatorysignals that cause expression of said introduced DNAs; and

wherein said bacterium produces an elevated amount of α-keto-butyratecompared to that in a corresponding wild-type bacterium not comprisingsaid introduced DNAs.

A bacterium, the genome of which comprises introduced DNAs encoding thefollowing enzymes:

a wild-type or deregulated aspartate kinase;

homoserine dehydrogenase; and

a wild-type or deregulated threonine deaminase;

wherein said introduced DNAs are operatively linked to regulatorysignals that cause expression of said introduced DNAs; and

wherein said bacterium produces an increased amount of α-keto-butyratecompared to that in a wild-type bacterium not comprising said introducedDNAs.

A bacterium, the genome of which comprises introduced DNAs encoding thefollowing enzymes:

a wild-type or deregulated aspartate kinase;

homoserine dehydrogenase;

threonine synthase; and

a wild-type or deregulated threonine deaminase;

wherein said introduced DNAs are operatively linked to regulatorysignals that cause expression of said introduced DNAs; and

wherein said bacterium produces an increased amount of α-keto-butyratecompared to that in a wild-type bacterium not comprising said introducedDNAs.

A bacterium, the genome of which comprises introduced DNAs encoding thefollowing enzymes:

a wild-type or deregulated threonine deaminase; and

an α-ketoacid decarboxylase E1 subunit that exhibits improved bindingand decarboxylating activities with α-ketobutyrate compared withpyruvate decarboxylase E1 subunit naturally present in pyruvatedehydrogenase complex of said bacterium;

wherein each of said introduced DNAs is operatively linked to regulatorysignals that cause expression of said introduced DNAs; and

wherein said bacterium produces an elevated amount of propionyl-CoAcompared to that in a corresponding wild-type bacterium not comprisingsaid introduced DNAs.

The foregoing bacterium, wherein said α-ketoacid decarboxylase E1subunit that exhibits improved binding and decarboxylating activitieswith α-ketobutyrate is a branched chain α-ketoacid decarboxylase E1subunit such as that from bovine kidney, Pseudomonas putida, or Bacillussubtilis.

A bacterium, the genome of which comprises introduced DNAs encoding:

a wild-type or deregulated threonine deaminase;

a pyruvate decarboxylase E1 subunit that exhibits improved binding anddecarboxylating activities with α-ketobutyrate compared with pyruvatedecarboxylase E1 subunit naturally present in pyruvate dehydrogenasecomplex in said bacterium; and

a dihydrolipoyl transacylase E2 subunit exhibiting improved transacylaseactivity with α-ketobutyrate compared with dihydrolipoyl transacetylaseE2 subunit naturally present in pyruvate dehydrogenase complex in saidbacterium;

wherein said introduced DNAs are operatively linked to regulatorysignals that cause expression of said introduced DNAs; and

wherein said bacterium produces an elevated amount of propionyl-CoAcompared to that in a corresponding wild-type bacterium not comprisingsaid introduced DNAs.

The foregoing bacterium, wherein said α-ketoacid decarboxylase E1subunit that exhibits improved binding and decarboxylating activitieswith α-ketobutyrate is a branched chain α-ketoacid decarboxylase E1subunit such as that from bovine kidney, Pseudomonas putida, or Bacillussubtilis, and said dihydrolipoyl transacylase E2 subunit exhibitingimproved transacylase activity with α-ketobutyrate is one such as thatfrom bovine kidney, Pseudomonas putida, or Bacillus subtilis.

A bacterium, the genome of which comprises introduced DNAs encoding thefollowing enzymes:

a wild-type or deregulated threonine deaminase; and

pyruvate oxidase;

wherein said introduced DNAs are operably linked to regulatory signalsthat cause expression of said introduced DNAs; and

wherein said bacterium produces an elevated amount of propionyl-CoAcompared that in a corresponding wild-type bacterium not comprising saidintroduced DNAs.

A bacterium, the genome of which comprises introduced DNAs encoding thefollowing enzymes:

threonine synthase;

a wild-type or deregulated threonine deaminase; and

pyruvate oxidase;

wherein said introduced DNAs are operably linked to regulatory signalsthat cause expression of said introduced DNAs; and

wherein said bacterium produces an elevated amount of propionyl-CoAcompared that in a corresponding wild-type bacterium not comprising saidintroduced DNAs.

The foregoing bacterium, wherein said pyruvate oxidase is encoded by E.coli poxB.

A bacterium, the genome of which comprises introduced DNAs encoding thefollowing enzymes:

a wild-type or deregulated threonine deaminase;

pyruvate oxidase;

a β-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA and a β-ketothiolase capable of condensingacetyl-CoA and propionyl-CoA to produce β-ketovaleryl-CoA; or

a β-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA, and capable of condensing acetyl-CoA andpropionyl-CoA to produce β-ketovaleryl-CoA;

a β-ketoacyl-CoA reductase capable of reducing acetoacetyl-CoA andβ-ketovaleryl-CoA to produce β-hydroxybutyryl-CoA andβ-hydroxy-valeryl-CoA, respectively; and

a polyhydroxyalkanoate synthase capable of incorporatingβ-hydroxybutyryl-CoA and β-hydroxyvaleryl-CoA into P(3HB-co-3HV)copolymer;

wherein said introduced DNAs are operatively linked to regulatorysignals that cause expression of said introduced DNAs; and

wherein said bacterium produces P(3HB-co-3HV) copolymer.

A bacterium, the genome of which comprises introduced DNAs encoding thefollowing enzymes:

threonine synthase;

a wild-type or deregulated threonine deaminase;

pyruvate oxidase;

a β-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA and a β-ketothiolase capable of condensingacetyl-CoA and propionyl-CoA to produce β-ketovaleryl-CoA; or

a β-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA, and capable of condensing acetyl-CoA andpropionyl-CoA to produce β-ketovaleryl-CoA;

a β-ketoacyl-CoA reductase capable of reducing acetoacetyl-CoA andβ-ketovaleryl-CoA to produce β-hydroxybutyryl-CoA andβ-hydroxy-valeryl-CoA, respectively; and

a polyhydroxyalkanoate synthase capable of incorporatingβ-hydroxybutyryl-CoA and β-hydroxyvaleryl-CoA into P(3HB-co-3HV)copolymer;

wherein said introduced DNAs are operatively linked to regulatorysignals that cause expression of said introduced DNAs; and

wherein said bacterium produces P(3HB-co-3HV) copolymer.

A bacterium, the genome of which comprises introduced DNAs encoding thefollowing enzymes:

a wild-type or deregulated threonine deaminase;

a β-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA and a β-ketothiolase capable of condensingacetyl-CoA and propionyl-CoA to produce β-ketovaleryl-CoA; or

a β-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA, and capable of condensing acetyl-CoA andpropionyl-CoA to produce β-ketovaleryl-CoA;

a β-ketoacyl-CoA reductase capable of reducing acetoacetyl-CoA andβ-ketovaleryl-CoA to produce β-hydroxybutyryl-CoA andβ-hydroxy-valeryl-CoA, respectively; and

a polyhydroxyalkanoate synthase capable of incorporatingβ-hydroxybutyryl-CoA and β-hydroxyvaleryl-CoA into P(3HB-co-3HV)copolymer;

wherein said introduced DNAs are operatively linked to regulatorysignals that cause expression of said introduced DNAs; and

wherein said bacterium produces P(3HB-co-3HV) copolymer.

A bacterium, the genome of which comprises introduced DNAs encoding thefollowing enzymes:

threonine synthase;

a wild-type or deregulated threonine deaminase;

a β-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA and a β-ketothiolase capable of condensingacetyl-CoA and propionyl-CoA to produce β-ketovaleryl-CoA; or

a β-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA, and capable of condensing acetyl-CoA andpropionyl-CoA to produce β-ketovaleryl-CoA;

a β-ketoacyl-CoA reductase capable of reducing acetoacetyl-CoA andβ-ketovaleryl-CoA to produce β-hydroxybutyryl-CoA andβ-hydroxy-valeryl-CoA, respectively; and

a polyhydroxyalkanoate synthase capable of incorporatingβ-hydroxybutyryl-CoA and β-hydroxyvaleryl-CoA into P(3HB-co-3HV)copolymer;

wherein said introduced DNAs are operatively linked to regulatorysignals that cause expression of said introduced DNAs; and

wherein said bacterium produces P(3HB-co-3HV) copolymer.

The foregoing bacterium, wherein said β-ketothiolase capable ofcondensing acetyl-CoA and propionyl-CoA to produce β-ketovaleryl-CoA isBktB.

The foregoing bacterium, wherein said β-ketoacyl-CoA reductase isacetoacetyl-CoA reductase.

The foregoing bacterium, wherein said polyhydroxyalkanoate synthase isobtainable from a microorganism selected from the group consisting ofAlcaligenes eutrophus, Alcaligenes faecalis, Aphanothece sp.,Azotobacter vinelandii, Bacillus cereus, Bacillus megaterium,Beijerinkia indica, Derxia gummosa, Methylobacterium sp., Microcoleussp., Nocardia corallina, Pseudomonas cepacia, Pseudomonas extorquens,Pseudomonas oleovorans, Rhodobacter sphaeroides, Rhodobacter capsulatus,Rhodospirillum rubrum, and Thiocapsa pfennigii.

A bacterium, the genome of which comprises introduced DNA encoding awild-type or deregulated threonine deaminase;

wherein said introduced DNA is operatively linked to regulatory signalsthat cause expression of said introduced DNA; and

wherein said bacterium produces an increased amount of α-keto-butyratecompared to that in a corresponding wild-type bacterium not comprisingsaid introduced DNA.

A bacterium, the genome of which comprises introduced DNAs encoding thefollowing enzymes:

threonine synthase; and

a wild-type or deregulated threonine deaminase;

wherein said introduced DNAs are operatively linked to regulatorysignals that cause expression of said introduced DNAs; and

wherein said bacterium produces an increased amount of α-keto-butyratecompared to that in a corresponding wild-type bacterium not comprisingsaid introduced DNAs.

A bacterium, the genome of which comprises introduced DNAs encoding thefollowing enzymes:

a wild-type or deregulated aspartate kinase;

homoserine dehydrogenase;

a wild-type or deregulated threonine deaminase; and

pyruvate oxidase;

wherein said introduced DNAs are operatively linked to regulatorysignals that cause expression of said introduced DNAs; and

wherein said bacterium produces an increased amount of propionyl-CoAcompared to that in a corresponding, wild-type bacterium not comprisingsaid introduced DNAs.

A bacterium, the genome of which comprises introduced DNAs encoding thefollowing enzymes:

a wild-type or deregulated aspartate kinase;

homoserine dehydrogenase;

threonine synthase;

a wild-type or deregulated threonine deaminase; and

pyruvate oxidase;

wherein said introduced DNAs are operatively linked to regulatorysignals that cause expression of said introduced DNAs; and

wherein said bacterium produces an increased amount of propionyl-CoAcompared to that in a corresponding, wild-type bacterium not comprisingsaid introduced DNAs.

A bacterium, the genome of which comprises introduced DNAs encoding thefollowing enzymes:

a wild-type or deregulated aspartate kinase;

homoserine dehydrogenase;

a wild-type or deregulated threonine deaminase;

pyruvate oxidase; and

an acyl-CoA synthetase;

wherein said introduced DNAs are operatively linked to regulatorysignals that cause expression of said introduced DNAs; and

wherein said bacterium produces an increased amount of propionyl-CoAcompared to that in a corresponding, wild-type bacterium not comprisingsaid introduced DNAs.

A bacterium, the genome of which comprises introduced DNAs encoding thefollowing enzymes:

a wild-type or deregulated aspartate kinase;

homoserine dehydrogenase;

threonine synthase;

a wild-type or deregulated threonine deaminase;

pyruvate oxidase; and

an acyl-CoA synthetase;

wherein said introduced DNAs are operatively linked to regulatorysignals that cause expression of said introduced DNAs; and

wherein said bacterium produces an increased amount of propionyl-CoAcompared to that in a corresponding, wild-type bacterium not comprisingsaid introduced DNAs.

A bacterium, the genome of which comprises introduced DNAs encoding thefollowing enzymes:

a wild-type or deregulated threonine deaminase; and

an α-ketoacid decarboxylase E1 subunit that exhibits improved bindingand decarboxylating activities with α-ketobutyrate compared withpyruvate decarboxylase E1 subunit naturally present in pyruvatedehydrogenase complex in said bacterium;

wherein said introduced DNAs are operatively linked to regulatorysignals that cause expression of said introduced DNAs; and

wherein said bacterium produces an increased amount of propionyl-Co Acompared to that in a corresponding, wild-type bacterium not comprisingsaid introduced DNAs.

A bacterium, the genome of which comprises introduced DNAs encoding thefollowing enzymes:

threonine synthase;

a wild-type or deregulated threonine deaminase; and

an α-ketoacid decarboxylase E1 subunit that exhibits improved bindingand decarboxylating activities with α-ketobutyrate compared withpyruvate decarboxylase E1 subunit naturally present in pyruvatedehydrogenase complex in said bacterium;

wherein said introduced DNAs are operatively linked to regulatorysignals that cause expression of said introduced DNAs; and

wherein said bacterium produces an increased amount of propionyl-Co Acompared to that in a corresponding, wild-type bacterium not comprisingsaid introduced DNAs.

A bacterium, the genome of which comprises introduced DNAs encoding thefollowing enzymes:

a wild-type or deregulated aspartate kinase;

a wild-type or deregulated threonine deaminase; and

an α-ketoacid decarboxylase E1 subunit that exhibits improved bindingand decarboxylating activities with α-ketobutyrate compared withpyruvate decarboxylase E1 subunit naturally present in pyruvatedehydrogenase complex in said bacterium;

wherein said introduced DNAs are operatively linked to regulatorysignals that cause expression of said introduced DNAs; and

wherein said bacterium produces an increased amount of propionyl-CoAcompared to that in a corresponding, wild-type bacterium not comprisingsaid introduced DNAs.

A bacterium, the genome of which comprises introduced DNAs encoding thefollowing enzymes:

a wild-type or deregulated aspartate kinase;

threonine synthase;

a wild-type or deregulated threonine deaminase; and

an α-ketoacid decarboxylase E1 subunit that exhibits improved bindingand decarboxylating activities with α-ketobutyrate compared withpyruvate decarboxylase E1 subunit naturally present in pyruvatedehydrogenase complex in said bacterium;

wherein said introduced DNAs are operatively linked to regulatorysignals that cause expression of said introduced DNAs; and

wherein said bacterium produces an increased amount of propionyl-CoAcompared to that in a corresponding, wild-type bacterium not comprisingsaid introduced DNAs.

A bacterium, the genome of which comprises introduced DNAs encoding thefollowing enzymes:

a wild-type or deregulated aspartate kinase;

homoserine dehydrogenase;

a wild-type or deregulated threonine deaminase; and

an α-ketoacid decarboxylase E1 subunit that exhibits improved bindingand decarboxylating activities with α-ketobutyrate compared withpyruvate decarboxylase E1 subunit naturally present in pyruvatedehydrogenase complex in said bacterium;

wherein said introduced DNAs are operatively linked to regulatorysignals that cause expression of said introduced DNAs; and

wherein said bacterium produces an increased amount of propionyl-CoAcompared to that in a corresponding, wild-type bacterium not comprisingsaid introduced DNAs.

A bacterium, the genome of which comprises introduced DNAs encoding thefollowing enzymes:

a wild-type or deregulated aspartate kinase;

homoserine dehydrogenase;

threonine synthase;

a wild-type or deregulated threonine deaminase; and

an α-ketoacid decarboxylase E1 subunit that exhibits improved bindingand decarboxylating activities with α-ketobutyrate compared withpyruvate decarboxylase E1 subunit naturally present in pyruvatedehydrogenase complex in said bacterium;

wherein said introduced DNAs are operatively linked to regulatorysignals that cause expression of said introduced DNAs; and

wherein said bacterium produces an increased amount of propionyl-CoAcompared to that in a corresponding, wild-type bacterium not comprisingsaid introduced DNAs.

A bacterium, the genome of which comprises introduced DNAs encoding thefollowing enzymes:

a wild-type or deregulated threonine deaminase;

pyruvate oxidase; and

an α-ketoacid decarboxylase E1 subunit that exhibits improved bindingand decarboxylating activities with α-ketobutyrate compared withpyruvate decarboxylase E1 subunit naturally present in pyruvatedehydrogenase complex in said bacterium;

wherein said introduced DNAs are operatively linked to regulatorysignals that cause expression of said introduced DNAs; and

wherein said bacterium produces an increased amount of propionyl-CoAcompared to that in a corresponding, wild-type bacterium not comprisingsaid introduced DNAs.

A bacterium, the genome of which comprises introduced DNAs encoding thefollowing enzymes:

threonine synthase;

a wild-type or deregulated threonine deaminase;

pyruvate oxidase; and

an α-ketoacid decarboxylase E1 subunit that exhibits improved bindingand decarboxylating activities with α-ketobutyrate compared withpyruvate decarboxylase E1 subunit naturally present in pyruvatedehydrogenase complex in said bacterium;

wherein said introduced DNAs are operatively linked to regulatorysignals that cause expression of said introduced DNAs; and

wherein said bacterium produces an increased amount of propionyl-CoAcompared to that in a corresponding, wild-type bacterium not comprisingsaid introduced DNAs.

A bacterium, the genome of which comprises introduced DNAs encoding thefollowing enzymes:

a wild-type or deregulated threonine deaminase;

pyruvate oxidase;

an acyl-CoA synthetase; and

an α-ketoacid decarboxylase E1 subunit that exhibits improved bindingand decarboxylating activities with α-ketobutyrate compared withpyruvate decarboxylase E1 subunit naturally present in pyruvatedehydrogenase complex in said bacterium;

wherein said introduced DNAs are operatively linked to regulatorysignals that cause expression of said introduced DNAs; and

wherein said bacterium produces an increased amount of propionyl-CoAcompared to that in a corresponding, wild-type bacterium not comprisingsaid introduced DNAs.

A bacterium, the genome of which comprises introduced DNAs encoding thefollowing enzymes:

threonine synthase;

a wild-type or deregulated threonine deaminase;

pyruvate oxidase;

an acyl-CoA synthetase; and

an α-ketoacid decarboxylase E1 subunit that exhibits improved bindingand decarboxylating activities with α-ketobutyrate compared withpyruvate decarboxylase E1 subunit naturally present in pyruvatedehydrogenase complex in said bacterium;

wherein said introduced DNAs are operatively linked to regulatorysignals that cause expression of said introduced DNAs; and

wherein said bacterium produces an increased amount of propionyl-CoAcompared to that in a corresponding, wild-type bacterium not comprisingsaid introduced DNAs.

A bacterium, the genome of which comprises introduced DNAs encoding thefollowing enzymes:

a wild-type or deregulated aspartate kinase;

homoserine dehydrogenase;

a wild-type or deregulated threonine deaminase;

pyruvate oxidase; and

an α-ketoacid decarboxylase E1 subunit that exhibits improved bindingand decarboxylating activities with α-ketobutyrate compared withpyruvate decarboxylase E1 subunit naturally present in pyruvatedehydrogenase complex in said bacterium;

wherein said introduced DNAs are operatively linked to regulatorysignals that cause expression of said introduced DNAs; and

wherein said bacterium produces an increased amount of propionyl-CoAcompared to that in is a corresponding, wild-type bacterium notcomprising said introduced DNAs.

A bacterium, the genome of which comprises introduced DNAs encoding thefollowing enzymes:

a wild-type or deregulated aspartate kinase;

homoserine dehydrogenase;

threonine synthase;

a wild-type or deregulated threonine deaminase;

pyruvate oxidase; and

an α-ketoacid decarboxylase E1 subunit that exhibits improved bindingand decarboxylating activities with α-ketobutyrate compared withpyruvate decarboxylase E1 subunit naturally present in pyruvatedehydrogenase complex in said bacterium;

wherein said introduced DNAs are operatively linked to regulatorysignals that cause expression of said introduced DNAs; and

wherein said bacterium produces an increased amount of propionyl-CoAcompared to that in a corresponding, wild-type bacterium not comprisingsaid introduced DNAs.

A bacterium, the genome of which comprises introduced DNAs encoding thefollowing enzymes:

a wild-type or deregulated aspartate kinase;

homoserine dehydrogenase;

a wild-type or deregulated threonine deaminase;

pyruvate oxidase;

an acyl-CoA synthetase; and

an α-ketoacid decarboxylase E1 subunit that exhibits improved bindingand decarboxylating activities with α-ketobutyrate compared withpyruvate decarboxylase E1 subunit naturally present in pyruvatedehydrogenase complex in said bacterium;

wherein said introduced DNAs are operatively linked to regulatorysignals that cause expression of said introduced DNAs; and

wherein said bacterium produces an increased amount of propionyl-CoAcompared to that in a corresponding, wild-type bacterium not comprisingsaid introduced DNAs.

A bacterium, the genome of which comprises introduced DNAs encoding thefollowing enzymes:

a wild-type or deregulated aspartate kinase;

homoserine dehydrogenase;

threonine synthase;

a wild-type or deregulated threonine deaminase;

pyruvate oxidase;

an acyl-CoA synthetase; and

an α-ketoacid decarboxylase E1 subunit that exhibits improved bindingand decarboxylating activities with α-ketobutyrate compared withpyruvate decarboxylase E1 subunit naturally present in pyruvatedehydrogenase complex in said bacterium;

wherein said introduced DNAs are operatively linked to regulatorysignals that cause expression of said introduced DNAs; and

wherein said bacterium produces an increased amount of propionyl-CoAcompared to that in a corresponding, wild-type bacterium not comprisingsaid introduced DNAs.

A bacterium, the genome of which comprises introduced DNAs encoding thefollowing enzymes:

a wild-type or deregulated aspartate kinase;

a wild-type or deregulated threonine deaminase;

a β-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA and a β-ketothiolase capable of condensingacetyl-CoA and propionyl-CoA to produce β-ketovaleryl-CoA; or

β-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA, and capable of condensing acetyl-CoA andpropionyl-CoA to produce β-ketovaleryl-CoA;

a β-ketoacyl-CoA reductase capable of reducing acetoacetyl-CoA andβ-ketovaleryl-CoA to produce β-hydroxybutyryl-CoA andβ-hydroxy-valeryl-CoA, respectively; and

a polyhydroxyalkanoate synthase capable of incorporatingβ-hydroxybutyryl-CoA and β-hydroxyvaleryl-CoA into P(3HB-co-3HV)copolymer;

wherein said introduced DNAs are operatively linked to regulatorysignals that cause expression of said introduced DNAs; and

wherein said bacterium produces P(3HB-co-3HV) copolymer.

A bacterium, the genome of which comprises introduced DNAs encoding thefollowing enzymes:

a wild-type or deregulated aspartate kinase;

threonine synthase;

a wild-type or deregulated threonine deaminase;

a β-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA and a β-ketothiolase capable of condensingacetyl-CoA and propionyl-CoA to produce β-ketovaleryl-CoA; or

a β-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA, and capable of condensing acetyl-CoA andpropionyl-CoA to produce β-ketovaleryl-CoA;

a β-ketoacyl-CoA reductase capable of reducing acetoacetyl-CoA andβ-ketovaleryl-CoA to produce β-hydroxybutyryl-CoA andβ-hydroxy-valeryl-CoA, respectively; and

a polyhydroxyalkanoate synthase capable of incorporatingβ-hydroxybutyryl-CoA and β-hydroxyvaleryl-CoA into P(3HB-co-3HV)copolymer;

wherein said introduced DNAs are operatively linked to regulatorysignals that cause expression of said introduced DNAs; and

wherein said bacterium produces P(3HB-co-3HV) copolymer.

A bacterium, the genome of which comprises introduced DNAs encoding thefollowing enzymes:

a wild-type, partially, or totally lysine feedback inhibitioninsensitive aspartate kinase;

homoserine dehydrogenase;

a wild-type or deregulated threonine deaminase;

a β-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA and a β-ketothiolase capable of condensingacetyl-CoA and propionyl-CoA to produce β-ketovaleryl-CoA; or

a β-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA, and capable of condensing acetyl-CoA andpropionyl-CoA to produce β-ketovaleryl-CoA;

a β-ketoacyl-CoA reductase capable of reducing acetoacetyl-CoA andβ-ketovaleryl-CoA to produce β-hydroxybutyryl-CoA andβ-hydroxy-valeryl-CoA, respectively; and

a polyhydroxyalkanoate synthase capable of incorporatingβ-hydroxybutyryl-CoA and β-hydroxyvaleryl-CoA into P(3HB-co-3HV)copolymer;

wherein said introduced DNAs are operatively linked to regulatorysignals that cause expression of said introduced DNAs; and

wherein said bacterium produces P(3HB-co-3HV) copolymer.

A bacterium, the genome of which comprises introduced DNAs encoding thefollowing enzymes:

a wild-type, partially, or totally lysine feedback inhibitioninsensitive aspartate kinase;

homoserine dehydrogenase;

threonine synthase;

a wild-type or deregulated threonine deaminase;

a β-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA and a β-ketothiolase capable of condensingacetyl-CoA and propionyl-CoA to produce β-ketovaleryl-CoA; or

a β-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA, and capable of condensing acetyl-CoA andpropionyl-CoA to produce β-ketovaleryl-CoA;

a β-ketoacyl-CoA reductase capable of reducing acetoacetyl-CoA andβ-ketovaleryl-CoA to produce β-hydroxybutyryl-CoA andβ-hydroxy-valeryl-CoA, respectively; and

a polyhydroxyalkanoate synthase capable of incorporatingβ-hydroxybutyryl-CoA and β-hydroxyvaleryl-CoA into P(3HB-co-3HV)copolymer;

wherein said introduced DNAs are operatively linked to regulatorysignals that cause expression of said introduced DNAs; and

wherein said bacterium produces P(3HB-co-3HV) copolymer.

A bacterium, the genome of which comprises introduced DNAs encoding thefollowing enzymes:

a wild-type or deregulated threonine deaminase;

pyruvate oxidase;

a β-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA and a β-ketothiolase capable of condensingacetyl-CoA and propionyl-CoA to produce β-ketovaleryl-CoA; or

a β-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA, and capable of condensing acetyl-CoA andpropionyl-CoA to produce β-ketovaleryl-CoA;

a β-ketoacyl-CoA reductase capable of reducing acetoacetyl-CoA andβ-ketovaleryl-CoA to produce β-hydroxybutyryl-CoA andβ-hydroxy-valeryl-CoA, respectively; and

a polyhydroxyalkanoate synthase capable of incorporatingβ-hydroxybutyryl-CoA and β-hydroxyvaleryl-CoA into P(3HB-co-3HV)copolymer;

wherein said introduced DNAs are operatively linked to regulatorysignals that cause expression of said introduced DNAs; and

wherein said bacterium produces P(3HB-co-3HV) copolymer.

A bacterium, the genome of which comprises introduced DNAs encoding thefollowing enzymes:

threonine synthase;

a wild-type or deregulated threonine deaminase;

pyruvate oxidase;

a β-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA and a β-ketothiolase capable of condensingacetyl-CoA and propionyl-CoA to produce β-ketovaleryl-CoA; or

a β-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA, and capable of condensing acetyl-CoA andpropionyl-CoA to produce β-ketovaleryl-CoA;

a β-ketoacyl-CoA reductase capable of reducing acetoacetyl-CoA andβ-ketovaleryl-CoA to produce β-hydroxybutyryl-CoA andβ-hydroxy-valeryl-CoA, respectively; and

a polyhydroxyalkanoate synthase capable of incorporatingβ-hydroxybutyryl-CoA and β-hydroxyvaleryl-CoA into P(3HB-co-3HV)copolymer;

wherein said introduced DNAs are operatively linked to regulatorysignals that cause expression of said introduced DNAs; and

wherein said bacterium produces P(3HB-co-3HV) copolymer.

A bacterium, the genome of which comprises introduced DNAs encoding thefollowing enzymes:

a wild-type or deregulated threonine deaminase;

pyruvate oxidase;

an acyl-CoA synthetase;

a β-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA and a β-ketothiolase capable of condensingacetyl-CoA and propionyl-CoA to produce β-ketovaleryl-CoA; or

a β-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA, and capable of condensing acetyl-CoA andpropionyl-CoA to produce β-ketovaleryl-CoA;

a β-ketoacyl-CoA reductase capable of reducing acetoacetyl-CoA andβ-ketovaleryl-CoA to produce β-hydroxybutyryl-CoA andβ-hydroxy-valeryl-CoA, respectively; and

a polyhydroxyalkanoate synthase capable of incorporatingβ-hydroxybutyryl-CoA and β-hydroxyvaleryl-CoA into P(3HB-co-3HV)copolymer;

wherein said introduced DNAs are operatively linked to regulatorysignals that cause expression of said introduced DNAs; and

wherein said bacterium produces P(3HB-co-3HV) copolymer.

A bacterium, the genome of which comprises introduced DNAs encoding thefollowing enzymes:

threonine synthase;

a wild-type or deregulated threonine deaminase;

pyruvate oxidase;

an acyl-CoA synthetase;

a β-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA and a β-ketothiolase capable of condensingacetyl-CoA and propionyl-CoA to produce β-ketovaleryl-CoA; or

a β-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA, and capable of condensing acetyl-CoA andpropionyl-CoA to produce β-ketovaleryl-CoA;

a β-ketoacyl-CoA reductase capable of reducing acetoacetyl-CoA andβ-ketovaleryl-CoA to produce β-hydroxybutyryl-CoA andβ-hydroxy-valeryl-CoA, respectively; and

a polyhydroxyalkanoate synthase capable of incorporatingβ-hydroxybutyryl-CoA and β-hydroxyvaleryl-CoA into P(3HB-co-3HV)copolymer;

wherein said introduced DNAs are operatively linked to regulatorysignals that cause expression of said introduced DNAs; and

wherein said bacterium produces P(3HB-co-3HV) copolymer.

A bacterium, the genome of which comprises introduced DNAs encoding thefollowing enzymes:

a wild-type or deregulated aspartate kinase;

homoserine dehydrogenase;

a wild-type or deregulated threonine deaminase;

pyruvate oxidase;

a β-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA and a β-ketothiolase capable of condensingacetyl-CoA and propionyl-CoA to produce β-ketovaleryl-CoA; or

a β-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA, and capable of condensing acetyl-CoA andpropionyl-CoA to produce β-ketovaleryl-CoA;

a β-ketoacyl-CoA reductase capable of reducing acetoacetyl-CoA andβ-ketovaleryl-CoA to produce β-hydroxybutyryl-CoA andβ-hydroxy-valeryl-CoA, respectively; and

a polyhydroxyalkanoate synthase capable of incorporatingβ-hydroxybutyryl-CoA and β-hydroxyvaleryl-CoA into P(3HB-co-3HV)copolymer;

wherein said introduced DNAs are operatively linked to regulatorysignals that cause expression of said introduced DNAs; and

wherein said bacterium produces P(3HB-co-3HV) copolymer.

A bacterium, the genome of which comprises introduced DNAs encoding thefollowing enzymes:

a wild-type or deregulated aspartate kinase;

homoserine dehydrogenase;

threonine synthase;

a wild-type or deregulated threonine deaminase;

pyruvate oxidase;

a β-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA and a β-ketothiolase capable of condensingacetyl-CoA and propionyl-CoA to produce β-ketovaleryl-CoA; or

a β-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA, and capable of condensing acetyl-CoA andpropionyl-CoA to produce β-ketovaleryl-CoA;

a β-ketoacyl-CoA reductase capable of reducing acetoacetyl-CoA andβ-ketovaleryl-CoA to produce β-hydroxybutyryl-CoA andβ-hydroxy-valeryl-CoA, respectively; and

a polyhydroxyalkanoate synthase capable of incorporatingβ-hydroxybutyryl-CoA and β-hydroxyvaleryl-CoA into P(3HB-co-3HV)copolymer;

wherein said introduced DNAs are operatively linked to regulatorysignals that cause expression of said introduced DNAs; and

wherein said bacterium produces P(3HB-co-3HV) copolymer.

A bacterium, the genome of which comprises introduced DNAs encoding thefollowing enzymes:

a wild-type or deregulated aspartate kinase;

homoserine dehydrogenase;

a wild-type or deregulated threonine deaminase;

pyruvate oxidase;

an acyl-CoA synthetase;

a β-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA and a β-ketothiolase capable of condensingacetyl-CoA and propionyl-CoA to produce β-ketovaleryl-CoA; or

a β-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA, and capable of condensing acetyl-CoA andpropionyl-CoA to produce β-ketovaleryl-CoA;

a β-ketoacyl-CoA reductase capable of reducing acetoacetyl-CoA andβ-ketovaleryl-CoA to produce β-hydroxybutyryl-CoA andβ-hydroxy-valeryl-CoA, respectively; and

a polyhydroxyalkanoate synthase capable of incorporatingβ-hydroxybutyryl-CoA and β-hydroxyvaleryl-CoA into P(3HB-co-3HV)copolymer;

wherein said introduced DNAs are operatively linked to regulatorysignals that cause expression of said introduced DNAs; and

wherein said bacterium produces P(3HB-co-3HV) copolymer.

A bacterium, the genome of which comprises introduced DNAs encoding thefollowing enzymes:

a wild-type or deregulated aspartate kinase;

homoserine dehydrogenase;

threonine synthase;

a wild-type or deregulated threonine deaminase;

pyruvate oxidase;

an acyl-CoA synthetase;

a β-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA and a β-ketothiolase capable of condensingacetyl-CoA and propionyl-CoA to produce β-ketovaleryl-CoA; or

a β-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA, and capable of condensing acetyl-CoA andpropionyl-CoA to produce β-ketovaleryl-CoA;

a β-ketoacyl-CoA reductase capable of reducing acetoacetyl-CoA andβ-ketovaleryl-CoA to produce β-hydroxybutyryl-CoA andβ-hydroxy-valeryl-CoA, respectively; and

a polyhydroxyalkanoate synthase capable of incorporatingβ-hydroxybutyryl-CoA and β-hydroxyvaleryl-CoA into P(3HB-co-3HV)copolymer;

wherein said introduced DNAs are operatively linked to regulatorysignals that cause expression of said introduced DNAs; and

wherein said bacterium produces P(3HB-co-3HV) copolymer.

A bacterium, the genome of which comprises introduced DNAs encoding thefollowing enzymes:

a wild-type or deregulated threonine deaminase;

an α-ketoacid decarboxylase E1 subunit that exhibits improved bindingand decarboxylating activities with α-ketobutyrate compared withpyruvate decarboxylase E1 subunit naturally present in pyruvatedehydrogenase complex in said bacterium;

a β-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA and a β-ketothiolase capable of condensingacetyl-CoA and propionyl-CoA to produce β-ketovaleryl-CoA; or

a β-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA, and capable of condensing acetyl-CoA andpropionyl-CoA to produce β-ketovaleryl-CoA;

a β-ketoacyl-CoA reductase capable of reducing acetoacetyl-CoA andβ-ketovaleryl-CoA to produce β-hydroxybutyryl-CoA andβ-hydroxy-valeryl-CoA, respectively; and

a polyhydroxyalkanoate synthase capable of incorporatingβ-hydroxybutyryl-CoA and β-hydroxyvaleryl-CoA into P(3HB-co-3HV)copolymer;

wherein said introduced DNAs are operatively linked to regulatorysignals that cause expression of said introduced DNAs; and

wherein said bacterium produces P(3HB-co-3HV) copolymer.

A bacterium, the genome of which comprises introduced DNAs encoding thefollowing enzymes:

threonine synthase;

a wild-type or deregulated threonine deaminase;

an α-ketoacid decarboxylase E1 subunit that exhibits improved bindingand decarboxylating activities with α-ketobutyrate compared withpyruvate decarboxylase E1 subunit naturally present in pyruvatedehydrogenase complex in said bacterium;

a β-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA and a β-ketothiolase capable of condensingacetyl-CoA and propionyl-CoA to produce β-ketovaleryl-CoA; or

a β-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA, and capable of condensing acetyl-CoA andpropionyl-CoA to produce β-ketovaleryl-CoA;

a β-ketoacyl-CoA reductase capable of reducing acetoacetyl-CoA andβ-ketovaleryl-CoA to produce β-hydroxybutyryl-CoA andβ-hydroxy-valeryl-CoA, respectively; and

a polyhydroxyalkanoate synthase capable of incorporatingβ-hydroxybutyryl-CoA and β-hydroxyvaleryl-CoA into P(3HB-co-3HV)copolymer;

wherein said introduced DNAs are operatively linked to regulatorysignals that cause expression of said introduced DNAs; and

wherein said bacterium produces P(3HB-co-3HV) copolymer.

A bacterium, the genome of which comprises introduced DNAs encoding thefollowing enzymes:

a wild-type or deregulated aspartate kinase;

a wild-type or deregulated threonine deaminase;

an α-ketoacid decarboxylase E1 subunit that exhibits improved bindingand decarboxylating activities with α-ketobutyrate compared withpyruvate decarboxylase E1 subunit naturally present in pyruvatedehydrogenase complex in said bacterium;

a β-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA and a β-ketothiolase capable of condensingacetyl-CoA and propionyl-CoA to produce β-ketovaleryl-CoA; or

a β-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA, and capable of condensing acetyl-CoA andpropionyl-CoA to produce β-ketovaleryl-CoA;

a β-ketoacyl-CoA reductase capable of reducing acetoacetyl-CoA andβ-ketovaleryl-CoA to produce β-hydroxybutyryl-CoA andβ-hydroxy-valeryl-CoA, respectively; and

a polyhydroxyalkanoate synthase capable of incorporatingβ-hydroxybutyryl-CoA and β-hydroxyvaleryl-CoA into P(3HB-co-3HV)copolymer;

wherein said introduced DNAs are operatively linked to regulatorysignals that cause expression of said introduced DNAs; and

wherein said bacterium produces P(3HB-co-3HV) copolymer.

A bacterium, the genome of which comprises introduced DNAs encoding thefollowing enzymes:

a wild-type or deregulated aspartate kinase;

threonine synthase;

a wild-type or deregulated threonine deaminase;

an α-ketoacid decarboxylase E1 subunit that exhibits improved bindingand decarboxylating activities with α-ketobutyrate compared withpyruvate decarboxylase E1 subunit naturally present in pyruvatedehydrogenase complex in said bacterium;

a β-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA and a β-ketothiolase capable of condensingacetyl-CoA and propionyl-CoA to produce β-ketovaleryl-CoA; or

a β-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA, and capable of condensing acetyl-CoA andpropionyl-CoA to produce β-ketovaleryl-CoA;

a β-ketoacyl-CoA reductase capable of reducing acetoacetyl-CoA andβ-ketovaleryl-CoA to produce β-hydroxybutyryl-CoA andβ-hydroxy-valeryl-CoA, respectively; and

a polyhydroxyalkanoate synthase capable of incorporatingβ-hydroxybutyryl-CoA and β-hydroxyvaleryl-CoA into P(3HB-co-3HV)copolymer;

wherein said introduced DNAs are operatively linked to regulatorysignals that cause expression of said introduced DNAs; and

wherein said bacterium produces P(3HB-co-3HV) copolymer.

A bacterium, the genome of which comprises introduced DNAs encoding thefollowing enzymes:

a wild-type or deregulated aspartate kinase;

homoserine dehydrogenase;

a wild-type or deregulated threonine deaminase;

an α-ketoacid decarboxylase E1 subunit that exhibits improved bindingand decarboxylating activities with α-ketobutyrate compared withpyruvate decarboxylase E1 subunit naturally present in pyruvatedehydrogenase complex in said bacterium;

a β-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA and a β-ketothiolase capable of condensingacetyl-CoA and propionyl-CoA to produce β-ketovaleryl-CoA; or

a β-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA, and capable of condensing acetyl-CoA andpropionyl-CoA to produce β-ketovaleryl-CoA;

a β-ketoacyl-CoA reductase capable of reducing acetoacetyl-CoA andβ-ketovaleryl-CoA to produce β-hydroxybutyryl-CoA andβ-hydroxy-valeryl-CoA, respectively; and

a polyhydroxyalkanoate synthase capable of incorporatingβ-hydroxybutyryl-CoA and β-hydroxyvaleryl-CoA into P(3HB-co-3HV)copolymer;

wherein said introduced DNAs are operatively linked to regulatorysignals that cause expression of said introduced DNAs; and

wherein said bacterium produces P(3HB-co-3HV) copolymer.

A bacterium, the genome of which comprises introduced DNAs encoding thefollowing enzymes:

a wild-type or deregulated aspartate kinase;

homoserine dehydrogenase;

threonine synthase;

a wild-type or deregulated threonine deaminase;

an α-ketoacid decarboxylase E1 subunit that exhibits improved bindingand decarboxylating activities with α-ketobutyrate compared withpyruvate decarboxylase E1 subunit naturally present in pyruvatedehydrogenase complex in said bacterium;

a β-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA and a β-ketothiolase capable of condensingacetyl-CoA and propionyl-CoA to produce β-ketovaleryl-CoA; or

a β-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA, and capable of condensing acetyl-CoA andpropionyl-CoA to produce β-ketovaleryl-CoA;

a β-ketoacyl-CoA reductase capable of reducing acetoacetyl-CoA andβ-ketovaleryl-CoA to produce β-hydroxybutyryl-CoA andβ-hydroxy-valeryl-CoA, respectively; and

a polyhydroxyalkanoate synthase capable of incorporatingβ-hydroxybutyryl-CoA and β-hydroxyvaleryl-CoA into P(3HB-co-3HV)copolymer;

wherein said introduced DNAs are operatively linked to regulatorysignals that cause expression of said introduced DNAs; and

wherein said bacterium produces P(3HB-co-3HV) copolymer.

A bacterium, the genome of which comprises introduced DNAs encoding thefollowing enzymes:

a wild-type or deregulated threonine deaminase;

pyruvate oxidase;

an α-ketoacid decarboxylase E1 subunit that exhibits improved bindingand decarboxylating activities with α-ketobutyrate compared withpyruvate decarboxylase E1 subunit naturally present in pyruvatedehydrogenase complex in said bacterium;

a β-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA and a β-ketothiolase capable of condensingacetyl-CoA and propionyl-CoA to produce β-ketovaleryl-CoA; or

a β-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA, and capable of condensing acetyl-CoA andpropionyl-CoA to produce β-ketovaleryl-CoA;

a β-ketoacyl-CoA reductase capable of reducing acetoacetyl-CoA andβ-ketovaleryl-CoA to produce β-hydroxybutyryl-CoA andβ-hydroxy-valeryl-CoA, respectively; and

a polyhydroxyalkanoate synthase capable of incorporatingβ-hydroxybutyryl-CoA and β-hydroxyvaleryl-CoA into P(3HB-co-3HV)copolymer;

wherein said introduced DNAs are operatively linked to regulatorysignals that cause expression of said introduced DNAs; and

wherein said bacterium produces P(3HB-co-3HV) copolymer.

A bacterium, the genome of which comprises introduced DNAs encoding thefollowing enzymes:

threonine synthase;

a wild-type or deregulated threonine deaminase;

pyruvate oxidase;

an α-ketoacid decarboxylase E1 subunit that exhibits improved bindingand decarboxylating activities with α-ketobutyrate compared withpyruvate decarboxylase E1 subunit naturally present in pyruvatedehydrogenase complex in said bacterium;

a β-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA and a β-ketothiolase capable of condensingacetyl-CoA and propionyl-CoA to produce β-ketovaleryl-CoA; or

a β-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA, and capable of condensing acetyl-CoA andpropionyl-CoA to produce β-ketovaleryl-CoA;

a β-ketoacyl-CoA reductase capable of reducing acetoacetyl-CoA andβ-ketovaleryl-CoA to produce β-hydroxybutyryl-CoA andβ-hydroxy-valeryl-CoA, respectively; and

a polyhydroxyalkanoate synthase capable of incorporatingβ-hydroxybutyryl-CoA and β-hydroxyvaleryl-CoA into P(3HB-co-3HV)copolymer;

wherein said introduced DNAs are operatively linked to regulatorysignals that cause expression of said introduced DNAs; and

wherein said bacterium produces P(3HB-co-3HV) copolymer.

A bacterium, the genome of which comprises introduced DNAs encoding thefollowing enzymes:

a wild-type or deregulated threonine deaminase;

pyruvate oxidase;

an acyl-CoA synthetase;

an α-ketoacid decarboxylase E1 subunit that exhibits improved bindingand decarboxylating activities with α-ketobutyrate compared withpyruvate decarboxylase E1 subunit naturally present in pyruvatedehydrogenase complex in said bacterium;

a β-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA and a β-ketothiolase capable of condensingacetyl-CoA and propionyl-CoA to produce β-ketovaleryl-CoA; or

a β-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA, and capable of condensing acetyl-CoA andpropionyl-CoA to produce β-ketovaleryl-CoA;

a β-ketoacyl-CoA reductase capable of reducing acetoacetyl-CoA andβ-ketovaleryl-CoA to produce β-hydroxybutyryl-CoA andβ-hydroxy-valeryl-CoA, respectively; and

a polyhydroxyalkanoate synthase capable of incorporatingβ-hydroxybutyryl-CoA and β-hydroxyvaleryl-CoA into P(3HB-co-3HV)copolymer;

wherein said introduced DNAs are operatively linked to regulatorysignals that cause expression of said introduced DNAs; and

wherein said bacterium produces P(3HB-co-3HV) copolymer.

A bacterium, the genome of which comprises introduced DNAs encoding thefollowing enzymes:

threonine synthase;

a wild-type or deregulated threonine deaminase;

pyruvate oxidase;

an acyl-CoA synthetase;

an α-ketoacid decarboxylase E1 subunit that exhibits improved bindingand decarboxylating activities with α-ketobutyrate compared withpyruvate decarboxylase E1 subunit naturally present in pyruvatedehydrogenase complex in said bacterium;

a β-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA and a β-ketothiolase capable of condensingacetyl-CoA and propionyl-CoA to produce β-ketovaleryl-CoA; or

a β-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA, and capable of condensing acetyl-CoA andpropionyl-CoA to produce β-ketovaleryl-CoA;

a β-ketoacyl-CoA reductase capable of reducing acetoacetyl-CoA andβ-ketovaleryl-CoA to produce β-hydroxybutyryl-CoA andβ-hydroxy-valeryl-CoA, respectively; and

a polyhydroxyalkanoate synthase capable of incorporatingβ-hydroxybutyryl-CoA and β-hydroxyvaleryl-CoA into P(3HB-co-3HV)copolymer;

wherein said introduced DNAs are operatively linked to regulatorysignals that cause expression of said introduced DNAs; and

wherein said bacterium produces P(3HB-co-3HV) copolymer.

A bacterium, the genome of which comprises introduced DNAs encoding thefollowing enzymes:

a wild-type or deregulated aspartate kinase;

homoserine dehydrogenase;

a wild-type or deregulated threonine deaminase;

pyruvate oxidase;

an α-ketoacid decarboxylase E1 subunit that exhibits improved bindingand decarboxylating activities with α-ketobutyrate compared withpyruvate decarboxylase E1 subunit naturally present in pyruvatedehydrogenase complex in said bacterium;

a β-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA and a β-ketothiolase capable of condensingacetyl-CoA and propionyl-CoA to produce β-ketovaleryl-CoA; or

a β-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA, and capable of condensing acetyl-CoA andpropionyl-CoA to produce β-ketovaleryl-CoA;

a β-ketoacyl-CoA reductase capable of reducing acetoacetyl-CoA andβ-ketovaleryl-CoA to produce β-hydroxybutyryl-CoA andβ-hydroxy-valeryl-CoA, respectively; and

a polyhydroxyalkanoate synthase capable of incorporatingβ-hydroxybutyryl-CoA and β-hydroxyvaleryl-CoA into P(3HB-co-3HV)copolymer;

wherein said introduced DNAs are operatively linked to regulatorysignals that cause expression of said introduced DNAs; and

wherein said bacterium produces P(3HB-co-3HV) copolymer.

A bacterium, the genome of which comprises introduced DNAs encoding thefollowing enzymes:

a wild-type or deregulated aspartate kinase;

homoserine dehydrogenase;

threonine synthase;

a wild-type or deregulated threonine deaminase;

pyruvate oxidase;

an α-ketoacid decarboxylase E1 subunit that exhibits improved bindingand decarboxylating activities with α-ketobutyrate compared withpyruvate decarboxylase E1 subunit naturally present in pyruvatedehydrogenase complex in said bacterium;

a β-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA and a β-ketothiolase capable of condensingacetyl-CoA and propionyl-CoA to produce β-ketovaleryl-CoA; or

a β-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA, and capable of condensing acetyl-CoA andpropionyl-CoA to produce β-ketovaleryl-CoA;

a β-ketoacyl-CoA reductase capable of reducing acetoacetyl-CoA andβ-ketovaleryl-CoA to produce β-hydroxybutyryl-CoA andβ-hydroxy-valeryl-CoA, respectively; and

a polyhydroxyalkanoate synthase capable of incorporatingβ-hydroxybutyryl-CoA and β-hydroxyvaleryl-CoA into P(3HB-co-3HV)copolymer;

wherein said introduced DNAs are operatively linked to regulatorysignals that cause expression of said introduced DNAs; and

wherein said bacterium produces P(3HB-co-3HV) copolymer.

A bacterium, the genome of which comprises introduced DNAs encoding thefollowing enzymes:

a wild-type or deregulated aspartate kinase;

homoserine dehydrogenase;

a wild-type or deregulated threonine deaminase;

pyruvate oxidase;

an acyl-CoA synthetase;

an α-ketoacid decarboxylase E1 subunit that exhibits improved bindingand decarboxylating, activities with α-ketobutyrate compared withpyruvate decarboxylase E1 subunit naturally present in pyruvatedehydrogenase complex in said bacterium;

a β-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA and a β-ketothiolase capable of condensingacetyl-CoA and propionyl-CoA to produce β-ketovaleryl-CoA; or

a β-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA, and capable of condensing acetyl-CoA andpropionyl-CoA to produce β-ketovaleryl-CoA;

a β-ketoacyl-CoA reductase capable of reducing acetoacetyl-CoA andβ-ketovaleryl-CoA to produce β-hydroxybutyryl-CoA andβ-hydroxy-valeryl-CoA, respectively; and

a polyhydroxyalkanoate synthase capable of incorporatingβ-hydroxybutyryl-CoA and β-hydroxyvaleryl-CoA into P(3HB-co-3HV)copolymer;

wherein said introduced DNAs are operatively linked to regulatorysignals that cause expression of said introduced DNAs; and

wherein said bacterium produces P(3HB-co-3HV) copolymer.

A bacterium, the genome of which comprises introduced DNAs encoding thefollowing enzymes:

a wild-type or deregulated aspartate kinase;

homoserine dehydrogenase;

threonine synthase;

a wild-type or deregulated threonine deaminase;

pyruvate oxidase;

an acyl-CoA synthetase;

an α-ketoacid decarboxylase E1 subunit that exhibits improved bindingand decarboxylating activities with α-ketobutyrate compared withpyruvate decarboxylase E1 subunit naturally present in pyruvatedehydrogenase complex in said bacterium;

a β-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA and a β-ketothiolase capable of condensingacetyl-CoA and propionyl-CoA to produce β-ketovaleryl-CoA; or

a β-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA, and capable of condensing acetyl-CoA andpropionyl-CoA to produce β-ketovaleryl-CoA;

a β-ketoacyl-CoA reductase capable of reducing acetoacetyl-CoA andβ-ketovaleryl-CoA to produce β-hydroxybutyryl-CoA andβ-hydroxy-valeryl-CoA, respectively; and

a polyhydroxyalkanoate synthase capable of incorporatingβ-hydroxybutyryl-CoA and β-hydroxyvaleryl-CoA into P(3HB-co-3HV)copolymer;

wherein said introduced DNAs are operatively linked to regulatorysignals that cause expression of said introduced DNAs; and

wherein said bacterium produces P(3HB-co-3HV) copolymer.

A method of producing P(3HB-co-3HV) copolymer, comprising growing any ofthe foregoing bacteria, the genome of which comprises introduced DNAsencoding a β-ketothiolase, a β-ketoacyl-CoA reductase, and apolyhydroxy-alkanoate synthase, and recovering saidP(3HB-co-3HV)copolymer produced thereby.

A bacterium, the genome of which comprises introduced DNAs encoding thefollowing enzymes:

a β-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA and a β-ketothiolase capable of condensingacetyl-CoA and propionyl-CoA to produce β-ketovaleryl-CoA; or

a β-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA, and capable of condensing acetyl-CoA andpropionyl-CoA to produce β-ketovaleryl-CoA;

a β-ketoacyl-CoA reductase capable of reducing acetoacetyl-CoA andβ-ketovaleryl-CoA to produce β-hydroxybutyryl-CoA andβ-hydroxy-valeryl-CoA, respectively; and

a polyhydroxyalkanoate synthase capable of incorporatingβ-hydroxybutyryl-CoA and β-hydroxyvaleryl-CoA into P(3HB-co-3HV)copolymer;

wherein said introduced DNAs are operatively linked to regulatorysignals that cause expression of said introduced DNAs; and

wherein said bacterium produces P(3HB-co-3HV) copolymer.

The foregoing bacterium can be one that overproduces threonine. Thegenome thereof can further comprise, in addition to introduced DNAsencoding the enzymes listed above, introduced DNA encoding a wild-typeor deregulated threonine deaminase. Alternatively, the foregoingbacterium can be one that produces propionate or propionyl-CoA at levelsuseful for producing P(3HB-co-3HV) copolymer.

A method of producing P(3HB-co-3HV) copolymer, comprising:

culturing any of the four foregoing mentioned bacteria under conditionsand for a time conducive to the formation of P(3HB-co-3HV) copolymer;and

recovering said P(3HB-co-3HV) copolymer produced thereby.

A bacterium, the genome of which comprises introduced DNAs encoding thefollowing enzymes:

a β-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA and a β-ketothiolase capable of condensingacetyl-CoA and propionyl-CoA to produce β-ketovaleryl-CoA; or

a β-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA, and capable of condensing acetyl-CoA andpropionyl-CoA to produce β-ketovaleryl-CoA;

a β-ketoacyl-CoA reductase capable of reducing acetoacetyl-CoA andβ-ketovaleryl-CoA to produce β-hydroxybutyryl-CoA andβ-hydroxy-valeryl-CoA, respectively; and

a polyhydroxyalkanoate synthase capable of incorporatingβ-hydroxybutyryl-CoA and β-hydroxyvaleryl-CoA into P(3HB-co-3HV)copolymer;

wherein said introduced DNAs are operatively linked to regulatorysignals that cause expression of said introduced DNAs; and

wherein said bacterium produces P(3HB-co-3HV) copolymer when grown on amedium comprising propionic acid.

A method of producing P(3HB-co-3HV) copolymer, comprising:

culturing the foregoing bacterium on a medium comprising propionic acidunder conditions and for a time conducive to the formation ofP(3HB-co-3HV) copolymer, and

recovering said P(3HB-co-3HV) copolymer produced thereby.

A bacterial cell containing P(3HB-co-3HV) copolymer, wherein saidP(3HB-co-3HV) copolymer is produced within said bacterial cell as aresult of expression of at least one DNA sequence introduced thereinthat encodes an enzyme necessary for P(3HB-co-3HV) copolymer synthesis.

The foregoing bacterial cell, wherein said at least one DNA sequence isselected from the group consisting of a DNA sequence encoding aβ-keto-thiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-Co-A, a DNA sequence encoding a β-ketothiolasecapable of condensing acetyl-CoA and propionyl-CoA to produceβ-ketovaleryl-CoA, a DNA sequence encoding a β-ketoacyl-CoA reductase, aDNA sequence encoding a polyhydroxyalkanoate synthase capable ofincorporating β-hydroxybutyryl-CoA and β-hydroxyvaleryl-CoA intoP(3HB-co-3HV) copolymer, and combinations thereof.

The foregoing bacterial cell, further comprising a DNA sequenceintroduced therein that encodes a wild-type or deregulated threoninedeaminase, wherein said DNA sequence encoding said wild-type orderegulated threonine deaminase is expressed.

A method for transforming canola, comprising:

(a) preparing a stem explant from a canola plant by:

(i) removing leaves and buds along the stem and removing 4-5 inches ofsaid stem below the flower buds; and

(ii) cutting said 4-5 inches of stem into segments;

(b) inserting DNA to be introduced into said explant of step (a) byinoculating said explant with a disarmed Agrobacterium tumefaciensvector containing said DNA;

(c) culturing said explant of step (b) in the basal-side downorientation;

(d) selecting transformed explant tissue; and

(e) regenerating a differentiated transformed plant from saidtransformed explant tissue of step (d).

A method for transforming soybean, comprising:

(a) preparing a cotyledon explant from a soybean seedling by:

(i) incubating said seedling at about 0° C. to about 10° C. for at least24 hours;

(ii) removing the hypocotyl region by cutting in the region of fromabout 0.2 to about 1.5 cm below the cotyledonary node;

(ii) splitting and completely separating the remaining attachedhypocotyl segment, also thereby separating the two cotyledons;

(iii) removing the epicotyl from the cotyledon to which it remainsattached; and

(iv) wounding the cotyledon in the region of said axillary bud;

(b) inserting DNA to be introduced into said explant of step (a) byinoculating at least the region adjacent to the axillary bud of theexplant with a disarmed Agrobacterium tumefaciens vector containing saidDNA;

(c) selecting transformed explant tissue; and

(d) regenerating a differentiated transformed plant from saidtransformed explant tissue of step (c).

A soybean explant prepared by steps (a)(i)-(a)(iv) of the precedingmethod.

Soybean tissue prepared from a seedling cotyledon pair containing anepicotyl, axillary buds, and hypocotyl tissue, comprising a singlecotyledon containing an axillary bud and associated hypocotyl segmentextending from about 0.2 to about 1.5 cm below the cotyledonary node;

wherein said associated hypocotyl segment is completely separated fromits adjacent hypocotyl segment attached to the remaining cotyledon, thusseparating said cotyledons;

wherein said epicotyl has been removed from the cotyledon to which it isattached;

wherein the cotyledon is wounded in the region of said axillary bud; and

wherein said seedling has been incubated at a temperature of from about0° C. to about 10° C. for at least about 24 hours prior to preparingsaid soybean tissue.

The present invention provides a novel means towards obtainingbiosynthetic hydroxyalkanoate polymers. In a preferred embodiment, thepolymers have a single mode molecular weight distribution. As usedherein, a single mode molecular weight distribution refers to a plot ofmolecular weight (x-axis) against population (y-axis), resulting in asingle peak. Multiple mode molecular weight distributions display two ormore peaks in such a plot. The plot is first ‘smoothed’ by generally anymathematically valid model, and preferably by the smoothing functiondescribed in “Exploratory Data Analysis” (Tukey, J. W. Addison-Wesleypublishers, Reading, Mass., 1977, pp 205-235). The three highest peakheights are selected, and their peak heights added together to form apeak sum. In order for a plot to represent a single mode molecularweight distribution, the highest peak height must comprise at leastabout 80% of the peak sum. Molecular weight may be determined by methodssuch as size exclusion chromatography or gel permeation chromatography.In a further preferred embodiment, the polymers have a small molecularweight distribution. As used herein, molecular weight distribution, alsoreferred to as polydispersity, is calculated by dividing the weightaverage molecular weight by the number average molecular weight.

In a preferred embodiment, a plant extract will contain apolyhydroxyalkanoate polymer wherein the polyhydroxyalkanoate polymerwas produced by a plant, and where the polyhydroxyalkanoate polymer hasa single mode molecular weight distribution. As used herein, a plantextract refers to materials prior to chromatographic separation. Theextract may be a plant lysate, or the materials dissolved fromcontacting the plant or plant materials with a suitable solventincluding, but not limited to alcohol or chloroform. Lysates may beprepared by a variety of methods including, but not limited tomechanical damage, chemical treatment, or enzymatic digestion. Thepolyhydroxyalkanoate polymer preferably has a molecular weightdistribution of between about 1 and about 5, preferably between about1.5 and about 4.5, more preferably between about 2 and about 4, and mostpreferably about 2.1 or about 2.5. The polyhydroxyalkanoate polymer maybe a homopolymer or a copolymer. The polyhydroxyalkanoate polymer maygenerally be any polyhydroxyalkanoate polymer compatible with theinventive processes, and more preferably a polymer of 3-hydroxybutyrate,3-hydroxyhexanoate, 3-hydroxyoctanoate, 3-hydroxydecanoate,3-hydroxydodecanoate, 3-hydroxytetradecanoate, 3-hydroxyhexadecanoate,3-hydroxyoctadecanoate, 3-hydroxyeicosanoate, 3-hydroxydocosanoate, orcopolymers thereof. In more preferred embodiments, the homopolymer ispoly(3-hydroxybutyrate) or poly(4-hydroxybutyrate), and the to copolymeris poly(3-hydroxybutyrate-co-3-hydroxyvalerate),poly(3-hydroxybutyrate-co-4-hydroxybutyrate),poly(3-hydroxybutyrate-co-3-hydroxyhexanoate),poly(4-hydroxybutyrate-co-3-hydroxyhexanoate), orpoly(3-hydroxybutyrate-co-4-hydroxybutyrate-co-3-hydroxyhexanoate). Theplant in which the polyhydroxyalkanoate polymer is biosynthesized isgenerally any plant suitable for the biosynthesis ofpolyhydroxyalkanoate polymers, and more preferably is tobacco, wheat, ispotato, Arabidopsis, corn, soybean, canola, oil seed rape, sunflower,flax, peanut, sugarcane, swtichgrass, or alfalfa. The number averagemolecular weight of the polyhydroxyalkanoate polymer is preferablygreater than about 100,000, more preferably greater than about 300,000,and most preferably greater than about 500,000.

The invention further provides methods for the preparation ofpolyhydroxyalkanoate polymers having a single mode molecular weightdistribution. A preferred embodiment comprises the steps of (a)inserting into a plant cell nucleic acid molecules comprising apolyhydroxyalkanoate synthesis pathway, preferably comprising aβ-ketoacyl reductase gene, a β-ketothiolase gene, and apolyhydroxyalkanoate synthase gene; (b) isolating a transformed plantcell; (c) regenerating the transformed plant cell to form a transformedplant; (d) selecting a transformed plant which produces apolyhydroxyalkanoate polymer having a single mode molecular weightdistribution; and (e) isolating the polyhydroxyalkanoate polymer. Theβ-ketoacyl reductase, β-ketothiolase, and polyhydroxyalkanoate synthasegenes may generally be of any source suitable for participation in theinventive processes, more preferably the genes are Alcaligeneseutrophus, Alcaligenes faecalis, Aphanothece sp., Azotobactervinelandii, Bacillus cereus, Bacillus megaterium, Beijerinkia indica,Derxia gummosa, Methylobacterium sp., Microcoleus sp., Nocardiacorallina, Pseudomonas cepacia, Pseudomonas extorquens, Pseudomonasoleovorans, Rhodobacter sphaeroides, Rhodobacter capsulatus,Rhodospirillum rubrum, or Thiocapsa pfennigii genes, and most preferablyare Alcaligenes eutrophus genes. The polyhydroxyalkanoate polymerpreferably has a molecular weight distribution of between about 1 andabout 5, preferably between about 1.5 and about 4.5, more preferablybetween about 2 and about 4, and most preferably about 2.1 or about 2.5.The polyhydroxyalkanoate polymer may be a homopolymer or a copolymer.The polyhydroxyalkanoate polymer may generally be anypolyhydroxyalkanoate polymer compatible with the inventive processes,and more preferably a polymer of 3-hydroxybutyrate, 3-hydroxyhexanoate,3-hydroxyoctanoate, 3-hydroxydecanoate, 3-hydroxydodecanoate,3-hydroxytetradecanoate, 3-hydroxyhexadecanoate, 3-hydroxyoctadecanoate,3-hydroxyeicosanoate, 3-hydroxydocosanoate, or copolymers thereof. Inmore preferred embodiments, the homopolymer is poly(3-hydroxybutyrate)or poly(4-hydroxybutyrate), and the copolymer ispoly(3-hydroxybutyrate-co-3-hydroxyvalerate),poly(3-hydroxybutyrate-co-4-hydroxybutyrate),poly(3-hydroxybutyrate-co-3-hydroxyhexanoate),poly(4-hydroxybutyrate-co-3-hydroxyhexanoate), orpoly(3-hydroxybutyrate-co-4-hydroxybutyrate-co-3-hydroxyhexanoate). Theplant in which the polyhydroxyalkanoate polymer is biosynthesized isgenerally any plant suitable for the biosynthesis ofpolyhydroxyalkanoate polymers, and more preferably is tobacco, wheat,potato, Arabidopsis, corn, soybean, canola, oil seed rape, sunflower,flax, peanut, sugarcane, swtichgrass, or alfalfa. The number averagemolecular weight of the polyhydroxyalkanoate polymer is preferablygreater than about 100,000, more preferably greater than about 300,000,and most preferably greater than about 500,000. The transformed plantcells and plants may further comprise nucleic acid molecules havinggenes that allow the plant to synthesize additional polyhydroxyalkanoateprecursors. In a preferred embodiment, the nucleic acid moleculesfurther comprise succinyl-CoA:acetyl-CoA CoA transferase, succinatesemialdehyde dehydrogenase, 4-hydroxybutyrate dehydrogenase, andhydroxybutyryl-CoA:acetyl-CoA CoA transferase genes. The inventivemethods may be further extended by including a step of generating ahomozygous daughter plant derived from the transformed plant.Conventional methods such as growing daughter plants from seeds, andcross-breeding may be used to generate such a homozygous daughter plant.The transformed plant may be analyzed to determine the copy number ofthe polyhydroxyalkanoate synthase gene. It is preferable that thesynthase gene be present in a low copy number, more preferably less thanfive, and most preferably present at a single copy. Copy number may bedetermined by any method known to those of skill in the art, andpreferably by Southern blotting.

The invention further provides methods for selecting a transformed plantcell or plant that is particularly suitable for the production of apolyhydroxyalkanoate polymer having a single mode molecular weightdistribution. A preferred embodiment comprises the steps of (a)obtaining transformed plant cells or transformed plants, (b) analyzingthe plant cells or plants for the presence of a polyhydroxyalkanoatesynthase gene, (c) determining the copy number of thepolyhydroxyalkanoate synthase gene, and (d) selecting a transformedplant cell or transformed plant having a single copy of thepolyhydroxyalkanoate synthase gene. Copy number may be determined by anymethod known to those of skill in the art, and preferably by Southernblotting.

Further scope of the applicability of the present invention will becomeapparent from the detailed description and drawings provided below.However, it should be understood that the detailed description andspecific examples, while indicating preferred embodiments of the presentinvention, are given by way of illustration only since various changesand modifications within the spirit and scope of the invention willbecome apparent to those skilled in the art from this detaileddescription.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other aspects, features, and advantages of the presentinvention will be better understood from the following detaileddescription taken in conjunction with the accompanying drawings, all ofwhich are given by way of illustration only and are not limitative ofthe present invention.

For the sake of brevity, a description of many of the common geneticelements present in each of the plasmid figures is not included in eachfigure legend. These genetic elements include Amp (ampicillin bacterialresistance); Spec or Spc/Str (spectinomycin bacterial resistance); Kanor Kan903 (kanamycin bacterial resistance); P-Sp6 (Sp6 RNA polymerasepromoter); P-T7 (T7 RNA polymerase promoter); ori-pUC; ori-327; andori-322 (each designation refers to the ColE1 plasmid origin of DNAreplication of E. coli); ori-M13 (M13 phage origin of replication);ori-pACYC (plasmid origin of DNA replication of E. coli); ptrc and ptac(IPTG-inducible E. coli Trp/Lac fusion promoters); G10 (T7 gene 10translational enhancing leader); precA (recA SOS responsive promoter);e35S, p7S, and P-FMV (promoters for plant expression); ARABSSU1A orPEPSPS-transit:2 (chloroplast transit peptides); E9 3′ and NOS 3′ (plantpolyadenylation/transcription termination signals); ori-V (Agrobacteriumorigin of replication).

FIG. 1 shows the biochemical steps involved in the production of PHBfrom acetyl-CoA catalyzed by the A. eutrophus PHB biosynthetic enzymes.

FIG. 2 shows the biochemical steps involved in the production ofP(3HB-co-3HV) copolymer from acetyl-CoA and propionyl-CoA catalyzed byPHA biosynthetic enzymes of A. eutrophus.

FIG. 3 summarizes the pathways discussed herein that are involved in theproduction of P(3HB-co-3HV) copolymer.

FIG. 4 shows the structure of pMON25659. pMON25659 contains thewild-type E. coli biosynthetic threonine deaminase (ilvA) gene derivedby PCR. The nucleotide sequence of the wild-type biosynthetic threoninedeaminase gene is shown in SEQ ID NO:1.

FIG. 5 shows the structure of pMON25660. pMON25660 was used for theoverexpression of the wild-type biosynthetic threonine deaminase (IlvA)from the ptrc promoter in E. coli.

FIG. 6 shows the structure of pMON25676. pMON25676 contains the sameBamHI fragment as is present in pMON25659. This plasmid was used forpreparation of ssDNA for in vitro mutagenesis.

FIG. 7 shows the structure of pMON25679. pMON25679 contains the ilvA219(L447F) gene encoding the mutant E. coli biosynthetic threoninedeaminase created by site-directed mutagenesis of the wild-typethreonine deaminase gene contained in plasmid pMON25676. The nucleotidesequence of the ilvA219 biosynthetic threonine deaminase gene is shownin SEQ ID NO:5.

FIG. 8 shows the structure of pMON25680. pMON25680 contains the ilvA466(L481F) gene encoding the mutant E. coli biosynthetic threoninedeaminase created by site-directed mutagenesis of the wild-typethreonine deaminase gene contained in plasmid pMON25676. The nucleotidesequence of the ilvA466 biosynthetic threonine deaminase gene is shownin SEQ ID NO:7.

FIG. 9 shows the structure of pMON25681. pMON25681 contains theilvA219/466 (L447F/L481F) gene encoding the mutant E. coli biosyntheticthreonine deaminase created by site-directed mutagenesis of thewild-type threonine deaminase gene contained in plasmid pMON25676. Thenucleotide sequence of the ilvA219/466 biosynthetic threonine deaminasegene is shown in SEQ ID NO:8.

FIG. 10 shows the structure of pMON25682. pMON25682 was used for theoverexpression in E. coli of the IlvA219 (L447F) mutant biosyntheticthreonine deaminase from the ptrc promoter.

FIG. 11 shows the structure of pMON25683. pMON25683 was used for theoverexpression in E. coli of the IlvA466 (L481F) mutant biosyntheticthreonine deaminase from the ptrc promoter.

FIG. 12 shows the structure of pMON25684. pMON25684 was used for theoverexpression of the IlvA219/466 (L447F/L481F) mutant biosyntheticthreonine deaminase in E. coli.

FIG. 13 shows the structure of pMON25663. pMON25663 was used fortransient expression of the wild-type E. coli threonine deaminase (IlvA)in tobacco protoplasts.

FIG. 14 shows the structure of pMON25686. pMON25686 was used fortransient e expression of the IlvA219 (L447F) mutant threonine deaminasein tobacco protoplasts.

FIG. 15 shows the structure of pMON25687. pMON25687 was used fortransient expression of the IlvA466 (L481F) mutant threonine deaminasein tobacco protoplasts.

FIG. 16 shows the structure of pMON25688. pMON25688 was used fortransient expression of the IlvA219/466 (L447F/L481F) mutant threoninedeaminase in tobacco protoplasts.

FIG. 17 shows the expression, confirmed by Western blot analysis, ofwild-type and mutant biosynthetic E. coli threonine deaminases intobacco protoplasts via electroporation with plasmids pMON26663,pMON25686, pMON25687, and pMON25688. Immunoreactive bands correspondingto the predicted molecular weight of each threonine deaminase (56 kDa)were observed in replicates, designated “1” and “2”.

FIG. 18 shows the structure of pMON25668. pMON25668 is the Agrobacteriumplant transformation plasmid used to test expression of the wild-type E.coli threonine deaminase (IlvA) in soybean callus. The phbA gene onplasmid pMON25668 was not required for the expression of threoninedeaminase.

FIG. 19 shows the expression and activity of wild-type E. colibiosynthetic threonine deaminase in transformed soybean callus.

FIG. 20 shows the structure of pMON25628. pMON25628 was used for theoverexpression in E. coli of the A. eutrophus PhbB reductase from theprecA promoter.

FIG. 21 shows the structure of pMON25629. pMON25629 was used for theoverexpression in E. coli of the A. eutrophus PhbC synthase from theptac promoter.

FIG. 22 shows the structure of pMON25636. pMON25636 was used for theoverexpression in E. coli of the A. eutrophus PhbA β-keto-thiolase fromthe ptrc promoter.

FIG. 23 shows the structure of pMON25626. pMON25626 was theAgrobacterium plant transformation vector containing the geneticelements for seed-specific, plastid-targeted expression of PhbBreductase and PhbC synthase used to transform canola and soybean.

FIG. 24 shows the structure of pMON25638. pMON25638 was theAgrobacterium plant transformation vector containing the geneticelements for seed specific, plastid-targeted expression of PhbAβ-keto-thiolase used to transform canola and soybean.

FIG. 25 shows the structure of pMON25822. This plasmid is derived fromthe broad host-range vector pBBR1MCS-3 (Kovach, 1994), and expresses A.euirophus bktB from the A. eutrophus bktB promoter. The bktB promoter isincluded on pMON25728, discussed in example 8.

FIG. 26 shows a GPC trace of polyhydroxyalkanoate polymers isolated fromthe transformed canola of Example 10.

FIG. 27 shows a GPC trace of polyhydroxyalkanoate polymers isolated fromtransformed soybean of Example 10.

Conventional methods of gene isolation, molecular cloning, vectorconstruction, etc., are well known in the art and are summarized inSambrook et al., 1989, and Ausubel et al., 1989. One skilled in the artcan readily reproduce the plasmids vectors described above without undueexperimentation employing these methods in conjunction with the cloninginformation provided by the figures attached hereto. The various DNAsequences, fragments, etc., necessary for this purpose can be readilyobtained as components of commercially available plasmids, or areotherwise well known in the art and publicly available.

The following detailed description of the invention is provided to aidthose skilled in the art in practicing the present invention. Even so,the following detailed description should not be construed to undulylimit the present invention as modifications and variations in theembodiments discussed herein can be made by those of ordinary skill inthe art without departing from the spirit or scope of the presentinventive discovery.

The references cited herein evidence the level of skill in the art towhich the present invention pertains. The contents of each of thesereferences, including the references cited therein, are hereinincorporated by reference in their entirety.

The following Examples describe a variety of different methods forenhancing the levels of threonine, α-ketobutyrate, and propionyl-CoA,the latter being a direct precursor substrate in the synthesis ofP(3HB-co-3HV) in bacteria and plants, and for producing PHA copolymersof differing C4/C5 compositions from various carbon sources.

Definitions

The following definitions are provided in order to aid those skilled inthe art in understanding the detailed description of the presentinvention.

“β-ketoacyl-CoA reductase” refers to a β-ketoacyl-CoA reducing enzymethat can convert a β-ketoacyl-CoA substrate to its correspondingβ-hydroxyacyl-CoA product using, for example, NADH or NADPH as thereducing cosubstrate. An example is the PhbB acetoacetyl-CoA reductaseof A. eutrophus.

“β-ketothiolase” refers to an enzyme that catalyzes the thiolyticcleavage of a β-ketoacyl-CoA, requiring free CoA, to form two acyl-CoAmolecules. However, the term “β-ketothiolase” as used herein also refersto enzymes that catalyze the condensation of two acyl-CoA molecules toform β-ketoacyl-CoA and free CoA, i.e., the reverse of the thiolyticcleavage reaction.

“CoA” refers to coenzyme A.

“C-terminal region” refers to the region of a peptide, polypeptide, orprotein chain from the middle thereof to the end that carries the aminoacid having a free a carboxyl group.

“Deregulated enzyme” refers to an enzyme that has been modified, forexample by mutagenesis, wherein the extent of feedback inhibition of thecatalytic activity of the enzyme by a metabolite is reduced such thatthe enzyme exhibits enhanced activity in the presence of said metabolitecompared to the unmodified enzyme. Some organisms possess deregulatedforms of such enzymes as the naturally occuring, wild-type form.

The term “encoding DNA” refers to chromosomal DNA, plasmid DNA, cDNA, orsynthetic DNA which codes on expression for any of the enzymes discussedherein.

The term “genome” as it applies to bacteria encompasses both thechromosome and plasmids within a bacterial host cell. Encoding DNAs ofthe present invention introduced into bacterial host cells can thereforebe either chromosomally-integrated or plasmid-localized. The term“genome” as it applies to plant cells encompasses not only chromosomalDNA found within the nucleus, but organelle DNA found within subcellularcomponents of the cell. DNAs of the present invention introduced intoplant cells can therefore be either chromosomally-integrated ororganelle-localized.

The terms “microbe” or “microorganism” refer to algae, bacteria, fungi,and protozoa.

The term “mutein” refers to a mutant form of a peptide, polypeptide, orprotein.

“N-terminal region” refers to the region of a peptide, polypeptide, orprotein chain from the amino acid having a free a amino group to themiddle of the chain.

“Overexpression” refers to the expression of a polypeptide or proteinencoded by a DNA introduced into a host cell, wherein said polypeptideor protein is either not normally present in the host cell, or whereinsaid polypeptide or protein is present in said host cell at a higherlevel than that normally expressed from the endogenous gene encodingsaid polypeptide or protein.

The term “plastid” refers to the class of plant cell organelles thatincludes amyloplasts, chloroplasts, chromoplasts, elaioplasts, eoplasts,etioplasts, leucoplasts, and proplastids. These organelles areself-replicating, and contain what is commonly referred to as the“chloroplast genome,” a circular DNA molecule that ranges in size fromabout 120 to about 217 kb, depending upon the plant species, and whichusually contains an inverted repeat region (Fosket, 1994).

The term “polyhydroxyalkanoate (PHA) synthase” refers to enzymes thatconvert β-hydroxyacyl-CoAs to polyhydroxyalkanoates and free CoA.

The term “u” refers to an enzyme unit. One unit catalyzes the productionof one μmole of product per minute.

EXAMPLE 1 Increased Production of Amino Acids in Bacterial and PlantCells Via Modification of the Activity of Aspartate Kinase, HomoserineDehydrogenase, and Threonine Synthase

Biosynthesis of the aspartate family of amino acids in plants occurs inthe plastids (Bryan, 1980). FIG. 3 shows the enzymatic steps involved inthe biosynthesis of P(3HB-co-3HV) copolymer from L-threonine viaα-keto-butyrate. Both acetyl-CoA and propionyl-CoA are precursorsubstrates in the synthesis of P(3HB-co-3HV) as shown in this figure.

The involvement of L-threonine in the formation of α-ketobutyrate isdescribed in detail below in Examples 2, 3, and 4. The present exampledescribes methods for increasing the available pools of L-threonine inbacterial and plant cells for P(3HB-co-3HV) copolymer biosynthesis.

Overexpression of a threonine or lysine deregulated aspartate kinase(AK), the enzyme that catalyzes the first step in the biosynthesis ofthreonine, lysine, and methionine (FIG. 3), results in an increase inthe intracellular levels of free L-threonine in the leaf by 55% (Shauland Galili, 1992a), and in the seed by 15-fold (Karchi et al., 1993).

A downstream enzyme in the aspartate pathway is dihydrodipicolinatesynthase (DHPS), which catalyzes the first committed step in lysinesynthesis (FIG. 3). Plants that overexpress a deregulated form of DHPSexhibit a 15-fold increase in free lysine (Shaul and Galili, 1992b).Plants expressing deregulated forms of both AK and DHPS exhibit adramatic increase in free lysine, but lower levels of free threonine(Shaul and Galili, 1993). Recently Falco et al., (1995) demonstratedincreased lysine in seeds of canola by 45% and in soybean by 33% byoverexpressing deregulated aspartate kinase and dihydrodipicolinatesynthase. Since both the AK and DHPS enzymes were overexpressed relativeto the wild-type enzymes, the biosynthetic pathway favored the formationof lysine over threonine (FIG. 3). In all cases, the amount of availableaspartate was not limiting in the formation of the end products.

In accordance with the present invention, threonine is an importantmetabolite in PHA biosynthesis as shown in FIG. 3. Overexpression ofeither a wild-type or deregulated aspartate kinase will increase theavailable pools of free threonine in the plastids. As discussed inExamples 2-4, below, overexpression of a deregulated threonine deaminasein bacterial or plant cells will greatly enhance threonine turnover toα-ketobutyrate. A possible effect of this enhanced threonine turnovermay be depletion of other aspartate family amino acids (see FIG. 3).Increasing the level of aspartate kinase can ameliorate this possiblenegative effect by increasing the pools of metabolic precursors involvedin the biosynthesis of lysine, threonine, and methionine. This willresult in enhancement of the free pools of lysine and methionine in thecell (i.e., in the plastids), thereby preventing starvation for lysineand/or methionine in the event that overexpression of threoninedeaminase causes such starvation. In addition to aspartate kinase (AK),homoserine dehydrogenase (HSD) and threonine synthase can be used toincrease further the levels of free threonine (see FIG. 3).

Deregulated aspartate kinases useful in the present invention canpossess a level of threonine and/or lysine insensitivity such that at0.1 mM threonine and/or 0.1 mM lysine and the Km concentration ofaspartate, the enzymes exhibit ≧10% activity relative to assayconditions in which threonine and/or lysine is absent. Deregulatedhomoserine dehydrogenases useful in the present invention preferablypossess a level of threonine insensitivity such that at 0.1 mM threonineand the Km concentration of aspartate semialdehyde, the enzymes exhibit≧10% activity relative to assay conditions in which threonine is absent.The Vmax values for the aspartate kinase and homoserine dehydrogenaseenzymes can fall within the range of 0.1-100 times that of theircorresponding wild-type enzymes. The Km values for the aspartate kinaseand homoserine dehydrogenase enzymes can fall within the range of0.01-10 times that of their corresponding wild-type enzymes.

Threonine synthase, the enzyme responsible for convertingphosphohomoserine to threonine, has been shown to enhance the level ofthreonine about 10-fold over the endogenous level when overexpressed inMethylobacillus glycogenes (Motoyama et al., 1994). In addition, E. colithreonine synthase overexpressed in tobacco cell culture resulted in a10-fold enhanced level of threonine from a six-fold increase in totalthreonine synthase activity (Muhitch, 1995). It is apparent from theseresults that overexpressed levels of threonine synthase in plants orother organisms will have the effect of increasing threonine levelstherein. This can be employed in the present invention to insure anenhanced supply of threonine for α-ketobutyrate and propionyl-CoAproduction, the latter by the action of the next enzyme, threoninedeaminase (described in Examples 2, 3, 4.). In conjunction with the PHAbiosynthetic enzymes described herein, threonine synthase can enhancethe formation of P(3HB-co-3HV) copolymer.

EXAMPLE 2 Increased Production of α-Ketobutyrate From L-Threonine ViaModified E. coli Biosynthetic Threonine Deaminases

L-threonine can be degraded by several metabolic routes (FIG. 3).Threonine aldolase (E.C. 4.1.2.5), a pyridoxal phosphate-containingenzyme, catalyzes threonine degradation to produce glycine andacetaldehyde, both of which can be metabolized to acetyl-CoA (Marcus andDekker, 1993). Acetyl-CoA is the precursor of the C4 monomer in PHB(FIG. 1). L-threonine 3-dehydrogenase (E.C. 1.1.1.103) convertsthreonine into aminoacetone and CO₂ (Boylan and Dekker, 1981).Alternatively, L-threonine can be converted to α-ketobutyrate bythreonine deaminase (also designated threonine dehydratase, E.C.4.2.1.16), as shown in FIG. 3. α-ketobutyrate is a direct metabolicprecursor of propionyl-CoA (FIG. 3). There is also competition forα-ketobutyrate in isoleucine biosynthesis (FIG. 3), and intransamination to produce 2-aminobutyrate (Shaner and Singh, 1993).

Biodegradative Threonine Deaminase

Threonine deaminase exists in two forms. One form is a biodegradativeenzyme, encoded by, for example, the tdcB gene of E. coli.Biodegradative threonine deaminase permits threonine to be used as acarbon source. The structural gene for this enzyme has been isolatedfrom E. coli K-12 (Goss and Datta, 1985), and the nucleotide sequencehas been determined (Datta et. al., 1987). The biodegradative form ofthreonine deaminase is allosterically activated by high levels of AMP(Shizuta and Hayashi, 1976), and is inactivated by glucose, pyruvate,and other metabolites (Feldman and Datta, 1975). Biodegradativethreonine deaminase is not feedback-inhibited by isoleucine. The K_(m)of E. coli biodegradative threonine deaminase for threonine has beendetermined to be 11 mM in the presence of activator (10 mM AMP) and 91mM in its absence (Shizuta and Tokushige, 1971). The V_(max) for theenzyme decreases by a factor of six in the absence of activators(Shizuta and Tokushige, 1971). Due to their requirement for activationby AMP to exhibit optimal activity in vivo, most degradative threoninedeaminases may not be optimal for purposes of the present invention.

Biosynthetic Threonine Deaminase

Another form of threonine deaminase is a biosynthetic enzyme thatcatalyzes the committed step in isoleucine biosynthesis (FIG. 3). In E.coli, this enzyme is encoded by the ilvA gene. It has recently beenreported (Colón et. al., 1995) that overexpression of the biosyntheticthreonine deaminase from C. glutamicum in C. lactofermentum leads to anincrease in the amount of isoleucine produced in the latter. In mostcases reported, the goal of utilizing an overexpressed threoninedeaminase was to increase the available pools of the essential aminoacid isoleucine.

Biosynthetic threonine deaminase was the first enzyme reported to befeedback inhibited by the endproduct of its biosynthetic pathway (i.e.,isoleucine), and served as a model for the development of the concept ofallosteric regulation (Changeux, 1961; Monod et. al., 1963).Biosynthetic threonine deaminase is strongly inhibited by isoleucine,and may not be optimal for producing enhanced levels of α-ketobutyratein its natural form, even when overexpressed in recombinant systems. Theresults reported in Table 3, infra, support this conclusion. Theseresults are discussed in greater detail in the section entitled“Biochemical Analysis of Wild-type and Mutant E. coli ThreonineDeaminases”, below.

In addition to being inhibited by isoleucine, biosynthetic threoninedeaminase exhibits kinetic positive cooperativity. This results in muchslower substrate turnover at low concentrations of threonine as comparedto an enzyme that behaves in a normal Michaelis-Menten manner (alsodiscussed in detail in the section entitled “Biochemical Analysis ofWild-type and Mutant E. coli Threonine Deaminases”, below). This too isan undesirable property for a threonine deaminase employed to producehigh levels of α-ketobutyrate.

Isoleucine Deregulated Mutants of Biosynthetic Threonine Deaminase

Isoleucine-deregulated mutants of biosynthetic threonine deaminase havebeen described in the literature in both bacteria and plants. Onemutant, isolated from S. typhimurium, is referred to as ilvA219 (LaRossaet. al., 1987). The ilvA219 mutation results from a change in amino acid447 from leucine (L) to phenylalanine (F). Anotherisoleucine-deregulated mutant of biosynthetic threonine deaminase,identified in E. coli, is referred to as ilvA466 (Taillon et al., 1988).The ilvA466 mutation results from a change in amino acid 481 fromleucine (L) to phenylalanine (F). A third isoleucine-deregulated mutantof threonine deaminase has been described in C. glutamicum, and resultsfrom a change in amino acid 323 from valine (V) to alanine (A) (Möckelet al., 1994).

There have been two published reports of isoleucine-deregulatedthreonine deaminases in plants (Strauss et al., 1985; Mourad and King,1995). In neither case was the mutation in the threonine deaminaseidentified.

Cloning and Expression of the Wild-type E. coli Biosynthetic ThreonineDeaminase Gene

Conventional molecular biological techniques for routine cloning,transformation, expression, etc., are well known to those of ordinaryskill in the art and are described in detail in Sambrook et al., 1989and Ausubel et al., 1989.

The nucleotide sequence of the wild-type E. coli ilvA threoninedeaminase gene is available in the Genbank database (accession numberK03503), and has also been published by Lawther et al. (1987). Thissequence is shown in SEQ ID NO:1.

In order to produce various isoleucine-deregulated mutant forms of E.coli threonine deaminase, the encoding genomic DNA was first isolatedfrom E. coli DH5α. Primers having the sequences shown in SEQ ID NO:2 andSEQ ID NO:3, respectively, were synthesized for use in the polymerasechain reaction (PCR) to amplify the gene encoding E. coli biosyntheticthreonine deaminase:

SEQ ID NO:2

TTTTTGGATCCGATATCTTAACCCGCCAAAAAGAACCTGAACGCCG

SEQ ID NO:3

TTTTTGGATCCATGGCTGACTCGCAACCCCTGTCCGG

The 1,573 base pair PCR fragment thus obtained was subcloned as a BamHIrestriction fragment into BamHI-digested plasmid pSP72 (Promega),creating pMON25659 (FIG. 4). pMON25659 was digested with NcoI, BstXI,and BamHI, and the two fragments containing the ilvA gene were purifiedand cloned as a triple ligation into NcoI and BamHI-digested pSE280(Invitrogen), creating plasmid pMON25660 (FIG. 5). Plasmid pMON25660contains the IPTG-inducible ptrc promoter fused to the wild-type E. colibiosynthetic threonine deaminase gene (ilvA) for expression in E. coli.

IlvA was expressed from plasmid pMON25660 by growing E. coli cellstransformed with pMON25660 by CaCl₂/heatshock treatment in Luria Broth(LB) at 37° C. to early log phase and inducing the cells with 0.5 mMIPTG. The addition of IPTG to the cells results in high leveltranscriptional activity from the ptrc promoter and expression of theIlvA.

IlvA protein extracts were prepared by pelleting induced cells,resuspending in 50 mM KPi and 5% glycerol, sonicating, and removingcellular debris by centrifugation. Extracts of E. coli containing IlvAwere employed for biochemical studies as discussed below in the sectionentitled “Biochemical Analysis of Wild-type and Mutant E. coli ThreonineDeaminases.”

IlvA was further purified by the addition of protamine sulfate to theprotein extract (3% final concentration) and incubation on ice for onehour. The precipitate was removed by centrifugation and discarded. Thesupernatant was brought to 20% ammonium sulfate concentration, incubatedat 4° C. for one hour, and the precipitate was removed by centrifugationand discarded. Ammonium sulfate was added to the supernatant to 50%final concentration, and the precipitate was pelleted by centrifugation.The pellet was resuspended in Buffer A: 10 mM Bis-Tris-Propane, pH7.0, 1mM EDTA, 1 mM DTT, and 1 mM isoleucine. The protein solution wasdialyzed (molecular weight cutoff of 12-14K) overnight in Buffer A. Thedialyzed solution was loaded onto a Q-sepharose fast flow column (2.5cm×40 cm, Pharmacia) pre-equilibrated with Buffer A. Elution ofthreonine deaminase was performed using 1250 ml Buffer A and 750 mlBuffer A plus 0.5M KCl (Buffer B). The elution gradient was increasedfrom 0% to 75% B over 200 min. The flow rate was 10 ml/min and fractionswere collected every 2.5 min. Active fractions were pooled and theenzyme precipitated by bringing the protein solution to 80% ammoniumsulfate saturation. Precipitated protein was pelleted by centrifugation,the pellet was dissolved in a small about of Buffer A, and dialyzedovernight against Buffer A plus 0.01 mM pyridoxal phosphate. Thedialyzed protein solution was loaded onto a Mono-Q 10/10 columnpre-equilibrated with Buffer A. Elution of threonine deaminase wasperformed using 400 ml Buffer A and 200 ml Buffer B in a gradient from0% to 70% B over 140 min. The flow rate was 4 ml/min and 2 min fractionswere collected. Active fractions with the highest specific activity wereanalyzed by SDS-PAGE/Coomassie Blue staining (data not shown). Thepurest fractions were pooled and used for the production of polyclonalantibodies in rabbits. The remainder of the protein was stored at −80°C.

Mutagenesis of the E. coli Biosynthetic Threonine Deaminase Gene

Single Replacement of Leucine With Phenylalanine at Amino Acid Position447

The cloned wild-type E. coli threonine deaminase gene (ilvA) (SEQ IDNO:1) was modified by substituting a leucine (L) codon for aphenylalanine (F) codon at amino acid position 447 as in the S.typhimurium ilvA219 enzyme (LaRossa et al., 1987) as follows.

The cloned E. coli ilvA threonine deaminase gene sequence in plasmidpMON25659 was excised as a BamHI fragment and cloned into BamHI-digestedpGEM-3zf(-) (Promega Corp.), creating pMON25676 (FIG. 6). PlasmidpMON25676 contains the M13 origin of replication for generation ofsingle strand DNA (ssDNA). Single strand DNA from plasmid pMON25676 wasprepared according to the pGEM® Single Strand systems procedure(Promega). The leucine codon corresponding to amino acid position 447was modified by site-directed mutagenesis using BioRad's Muta-Gene®phagemid in vitro mutagenesis protocol employing the following syntheticmutagenic oligonucleotide (SEQ ID NO:4):

SEQ ID NO:4

CAGCTTCGAGTTCCCGGAATCACCGGGCGCGTTCCTGCGCTTCC

Specificially, the use of this synthetic mutagenic oligonucleotidemodified SEQ ID NO:1 at nucleotide positions 1,317 (changing adenine toguanine, therefore removing an EcoRI restriction enzyme site), 1,339(changing cytosine to thymine), and 1,341 (changing guanine tocytosine). The combined substitutions at nucleotides 1,339 and 1,341changed the codon for amino acid 447 from one encoding leucine to oneencoding phenylalanine. The mutagenesis successfully recreated the S.typhimurium ilvA219 mutation in the E. coli biosynthetic threoninedeaminase. The removal of the EcoRI restriction enzyme site allowed forrapid screening of the resultant transformants following mutagenesis.Successful mutagenesis was confirmed by testing the putative mutants forinsensitivity to feedback inhibition by isoleucine, as discussed belowin the section entitled “Biochemical Analysis of Wild-type and Mutant E.coli Threonine Deaminases.”

The nucleotide sequence of the mutagenized E. coli biosyntheticthreonine deaminase gene containing the ilvA219 (L447F) mutation isshown in SEQ ID NO:5. This mutant gene is contained in plasmid pMON25679(FIG. 7).

Single Replacement of Leucine With Phenylalanine at Amino Acid Position481

The cloned wild-type E. coli threonine deaminase gene in pMON25676 wasmodified by site-directed mutagenesis by substituting a leucine (L)codon for a phenylalanine (F) codon at amino acid position 481 as in theE. coli IlvA466 enzyme (Taillon et al., 1988) using the mutagenicoligonucleotide of SEQ ID NO:6 as follows.

SEQ ID NO:6

TATCGCAGCCACGGCACCGACTACGGGOCGCTACTGGCGGCGTTCGAATTTGGCGACCATGAACC

Use of this synthetic mutagenic oligonucleotide changed the codon foramino acid 481 from one encoding leucine to one encoding phenylalanineby changing nucleotide 1,441 of SEQ ID NO:1 from cytosine to thymine.This substitution successfully recreated the E. coli ilvA466 (L481F)mutation. This mutagenic oligonucleotide also changed nucleotideposition 1,404 from thymine (T) to cytosine (C), thereby removing anNcoI restriction enzyme recognition site. The removal of the NcoIrestriction enzyme site allowed for rapid screening of the resultanttransformants following mutagenesis. Successful mutagenesis wasconfirmed by testing the putative mutants for insensitivity to feedbackinhibition by isoleucine as discussed below in the section entitled“Biochemical Analysis of Wild-type and Mutant E. coli ThreonineDeaminases.”

The nucleotide sequence of the mutagenized E. coli biosyntheticthreonine deaminase gene containing the ilvA466 (L481F) mutation isshown in SEQ ID NO:7. This mutant gene is contained in plasmid pMON25680(FIG. 8).

Replacement of Leucine With Phenylalanine at Amino Acid Positions 447and 481

Synthetic mutagenic oligonucleotides SEQ ID NO:4 and SEQ ID NO:6 wereused in concert to create a double E. coli mutant gene (L447F andL481F), referred to as ilvA219/466. The nucleotide sequence of thisdouble mutant gene is shown in SEQ ID NO:8. The removal of the NcoI andEcoRI restriction enzyme sites allowed for rapid screening of theresultant transformants following mutagenesis. Successful mutagenesiswas confirmed by testing the putative mutants for insensitivity tofeedback inhibition by isoleucine as discussed below in the sectionentitled “Biochemical Analysis of Wild-type and Mutant E. coli ThreonineDeaminases.” The IlvA219/466 double mutant gene is contained inpMON25681 (FIG. 9).

Cloning and Expression of the Mutant Threonine Deaminase Genes in E.coli

The plasmids pMON25679, pMON25680, and pMON25681 were separatelydigested with BstXI and BamHI, and the three mutant threonine deaminasegenes obtained thereby were cloned into BstXI and BamHI-digestedpMON25660 (FIG. 5). The resultant E. coli expression plasmids containedthe ptrc promoter fused to the mutant ilvA genes for overexpression inE. coli. The cloning resulted in the following plasmids: pMON25682 (FIG.10), containing the ilvA219 mutation (L447F); pMON25683 (FIG. 11),containing the ilvA466 mutation (L481F); and pMON25684 (FIG. 12),containing the ilvA219/466 mutations (L447F/L481F). Expression of allthe IlvA mutant enzymes was performed as described above in “Cloning andExpression of the Wild-type E. coli Biosynthetic Threonine Deaminase.”Extracts of E. coli containing the overexpressed mutant IlvAs wereassayed for insensitivity to feedback inhibition by isoleucine asdiscussed below in the section entitled “Biochemical Analysis ofWild-type and Mutant E. coli Threonine Deaminases.”

Biochemical Analysis of Wild-type and Mutant Threonine Deaminases

Kinetic analysis of the wild-type and mutant threonine deaminases wasperformed by determining threonine turnover catalyzed by these threoninedeaminases as assayed by the method of Burns (1971). Specifically,α-keto-butyrate production was monitored by coupling to L-lacticdehydrogenase (LDH)-catalyzed reduction of the α-ketoacid in thepresence of NADH to form (L)-2-hydroxybutyrate and NAD⁺. Thedisappearance of NADH absorbance at 340 nm provides a measure of thethreonine deaminase activity. Final reaction conditions were 100 mM KPi,pH 7.8, 0.20 mM NADH, 10 units/mL LDH (defined as LDH turnover ofα-ketobutyrate, not pyruvate), and variable threonine. R. rubrum wasgrown according to Brandl et al., 1989. The growth and induction of E.coli cells overexpressing the mutant and wild-type biosyntheticthreonine deaminases is described above. Cell extracts of R. rubrum andE. coli were prepared by sonicating pelleted cells (≈0.5 g from 100 mlof culture) in 2 ml of 100 mM KPi, pH 7.0, containing 5% (v/v) glycerol.After centrifugation to remove cellular debris, protein concentrationswere determined by the method of Bradford (1976), and threoninedeaminase activity was measured.

The kinetic parameters K_(m) and V_(max), and the level of positivecooperativity, i.e., the deviation from normal saturation kinetics whereat low substrate concentration the substrate turnover rate is less thana non-cooperative system, were determined. These values were found byfitting the rate data to the Hill equation (Equation 1), whichincorporates the coefficient n into the normal Michaelis-Menten equation(n=1 for a noncooperative system). An enzyme that displays no positivecooperativity exhibits an n value of ≦1.

v=V _(max) S ^(n)/(K _(m) +S)^(n)  (1)

In addition to the kinetic parameters, the degree of inhibition ofthreonine deaminase by isoleucine was also measured at fixed levels ofthreonine.

The results are shown in Tables 1 and 2.

TABLE 1 General Rate Effects of Isoleucine on Mutant and Wild-TypeThreonine Deaminases Threonine Threonine Deaminase Specific Activity,u/mg (%)¹ deaminase 0 mM Isoleucine 1.0 mM Isoleucine 10.0 mM IsoleucineE. coli wild-type 16.7 0.3 (<2) 0.3 (<2) E. coli L447F 16.0 16.3 (102)14.9 (93) E. coli L481F 20.3 11.3 (56) 0.3 (<2) E. coli L447F/481F 22.022.8 (103) 21.9 (99) R. rubrum wild-type 11.2 no data 10.0 (90) R.rubrum wild-type 1.2 no data 1.1 (90) (0.5 mM threonine) ¹Rates weredetermined using 10 mM threonine as substrate, except where indicated,at the indicated concentrations of isoleucine. Values in parenthesis are% activity relative to the results with 0 mM isoleucine.

As shown in Table 1, the E. coli IlvA219 enzyme (E. coli L447F mutant)is insensitive to isoleucine inhibition up to a concentration of 10 mMisoleucine. The E. coli IlvA466 enzyme (E. coli L481F mutant) exhibits amore modest level of insensitivity to inhibition by isoleucine,exhibiting 44% inhibition of enzyme activity at 1 mM isoleucine and 98%inhibition at 10 mM isoleucine. The novel double mutant enzyme (E. coliL447F/L481F mutant) is an improvement over the L447F mutant, exhibitingno inhibition at all in the presence of 10 mM isoleucine.

In addition to greatly enhanced insensitivity to isoleucine, the threemutant enzymes exhibit other enhanced kinetic properties. As shown inTable 2, the three mutant threonine deaminases exhibit lower K_(m)values and reduced or eliminated positive cooperativity compared to thewild-type enzyme. The latter is indicated by n values less than that ofwild-type E. coli threonine deaminase (Table 2). The combination of areduced n value and lower K_(m) can have a profound effect on the rateof substrate turnover, especially at low substrate concentrations. Forexample, using the reported K_(m) and n values for the threoninedeaminases reported in Table 2, with equivalent V_(max) values andthreonine=0.5 mM, the L447F mutant is approximately a factor of 10faster in substrate turnover than the wild-type enzyme.

TABLE 2 Kinetic Parameters for Mutant and Wild-type Threonine DeaminasesEnzyme¹ K_(m) Threonine (mM) Hill Coefficient (n) E coli wild-type 5 ± 11.5 ± 0.2 E. coli L447F 2.01 ± 0.06 0.91 ± 0.03 E. coli L481F 3.08 ±0.03 1.22 ± 0.01 E. coli L447F/L481F 2.15 ± 0.07 0.91 ± 0.04 R. rubrumwild-type 8.6 ± 0.3 0.79 ± 0.03 ¹The V_(max) for all threoninedeaminases are within a factor of two of each other, being approximately250-500 units/mg.

In vivo Analysis of Cloned E. coli ilvA Genes

In order to determine if overexpression of the wild-type E. colibiosynthetic threonine deaminase (IlvA) or an isoleucine-deregulatedthreonine deaminase mutant (for example, the IlvA466 (L481F) mutant E.coli threonine deaminase) could produce enhanced α-ketobutyrate levelsor P(3HB-co-3HV) copolymer from glucose and threonine in combinationwith the PHB biosynthetic operon of A. eutrophus, the followingexperiment was performed.

E. coli DH5α cells were transformed with pJM9238 (Kidwell et al., 1995)containing the PHB operon of A. eutrophus by CaCl₂/heat shock treatmentThe PHB operon was under the inducible control of of the ptac promoter.E. coli containing pJM9236 were used as a background control withrespect to threonine deaminase activity.

E. coli DH5α cells were also cotransformed by CaCl₂/heat shock treatmentwith pJM9238 and pMON25660 (FIG. 5) containing the wild-type E. colithreonine deaminase, under ptrc inducible control.

In a third experiment, E. coli DH5α cells were cotransformed withpJM9238 and pMON25683 (FIG. 11) containing the mutant E. coli ilvA466(L481F) threonine deaminase, under ptrc inducible control.

Transformed cells were grown on M9 minimal salts plus 0.2% gluconate and25 mM L-threonine. At early log phase, the cells were induced with IPTGto induce expression of all four enzymes (i.e., PhbA, PhbB, PhbC, andIlvA or IlvA466). Threonine deaminase activity was measured in cellextracts as described above in the section entitled “BiochemicalAnalysis of Wild-type and Mutant E. coli Threonine Deaminases.”intracellular α-ketobutyrate was isolated from approximately 50 mg (wetweight) of cells pelleted by centrifugation and then resuspended andsonicated in 1 ml of 100 mM KPi, pH 7.0, containing 5% glycerol (v/v).The level of α-ketobutyrate was determined by reverse-phase HPLCisolation of the dinitrophenylhydrazine derivative of the α-ketoacidaccording to Qureshi et al., (1982). Quantitation was based on peakintegration comparisons to a standard curve.

The PHA produced by the cells was extracted, hydrolyzed to themethylester, and analyzed by gas chromatography (GC) according to thefollowing procedure. The polymer was first extracted by centrifuging thecells at 7,000 rpm for 20 min., washing with 25 ml methanol,recentrifuging, washing with 15 ml hexane, and recentrifuging. Cellpellets were dried under N₂ for two hours, and the dry cell weightsdetermined. 6.5 ml of chloroform were added to extract the PHA at 100°C. for one hour. The solution was cooled and filtered through a PTFEsyringe filter (13 mm diameter, 0.45 μm pore). The PHA was precipitatedby the addition of 50 ml of methanol, collected by centrifugation, andthen washed with hexane. The polymer was dried at 70° C. for two hours,and after drying, the polymer weight was determined.

The recovered PHAs were subjected to methanolysis by the methoddescribed by Brandl et al., (1988), with some modifications. Three tofour milligrams of the polymer were dissolved in one ml chloroformcontaining a known amount (3.157 μmol/ml in a typical example) of methylbenzoate internal standard (Aldrich Chemical Company, USA) in a smallscrew-cap test tube. To this, 0.85 ml of methanol and 0.15 ml ofconcentrated sulfuric acid were added, and the mixture was refluxed at100° C. for 140 min. At the end of the reaction, 0.5 ml of deionizedwater was added and the tube vortexed for one min. After phaseseparation, the organic phase (bottom layer), which contains the methylesters of the constituent β-hydroxy-carboxylic acids from the PHAs, wasremoved and dried over sodium sulfate, and then used for analysis.

The methyl esters were assayed by gas chromatography in order todetermine the original copolymer composition, using a Varian 3400 gaschromatograph equipped with a DB-5 capillary column (0.25 mm×30 m, 0.25μfilm thickness; J&W Scientific, USA), a flame ionization detector, aVarian 8200 Autosampler, and a workstation with the Varian StarChromatography software. 1 μl of the sample was injected by splitinjection (split ratio of 50:1), using helium as the carrier gas (flowrate 2.5 ml/min). The temperatures of the injector and detector werekept at 250° C. A temperature program was used which separated theβ-hydroxybutyric acid and β-hydroxyvaleric acid methyl estersefficiently (60° C. for 3 min; temperature ramp of 12° C./min to 120°C.; temperature ramp of 30° C./min to 300° C.; 300° C. for 10 min).Under these conditions, the retention times of the methyl esters ofβ-hydroxybutyric acid and β-hydroxyvaleric acid were approximately 3.1min and 4.8 min, respectively, while that of the methyl benzoate was7.02 min. Quantitation was carried out based on standard curvesgenerated using methyl-3-(D)-hydroxybutyrate andmethyl-3-(D)-hydroxyvalerate (both obtained from Fluka, USA) as externalcalibration standards. In order to take into account the partitioning(Jan et al., 1995) of the methyl esters between the organic and aqueousphases, as well as to correct for the differences in partitioncoefficients of the respective methyl esters, the standards were treatedin a manner identical to that described above for the PHAs (except forthe omission of the reflux step) prior to generating the standardcurves.

The mol % C5 (3HV component) was determined in the isolated polymer. Theresults are shown in Table 3.

TABLE 3 Effect of Threonine Deaminase on α-Ketobutyrate Production and %C5 Content in PHBV³ Threonine Deaminase Specific Activity, u/mg (%)² %PHBV 0.1 mM [α-ketobutyrate] Dry Weight Tda¹ E. coli construct 0 mMIsoleucine Isoleucine μM (% C5) pJM9238⁴ <0.04 <0.04 13.4 35 (1.3)pJM9238/pMON25660⁵ 4.20 0.16 (3.8) 41.8 22 (1.1) pJM9238/pMON25683⁶ 0.590.56 (95) 579 27 (0.9) ¹Tda refers to threonine deaminase. ²Rates weredetermined using 10 mM threonine as substrate, at the indicatedconcentrations of isoleucine. Values in parenthesis are % activityrelative to the 0 mM isoleucine results. ³PHBV refers to P(3HB-co-3HV)copolymer. ⁴pJM9238 contains the A. eutrophus PHB operon. ⁵pMON25660contains the wild-type E. coli threonine deaminase. ⁶pMON25683 containsthe L481F mutant E. coli threonine deaminase.

Table 3 shows that there is about a three-fold increase inα-ketobutyrate concentration for the overexpressed wild-type threoninedeaminase compared to cells not overexpressing a threonine deaminase(41.8 μM vs. 13.4 μM α-keto-butyrate). This is evidently a reflection ofthe strong feedback regulation of the wild-type enzyme by isoleucine invivo. In contrast, the transformed cells containing the L481F mutantthreonine deaminase accumulated α-keto-butyrate to a level greater than40 times that in cells containing no overexpressed threonine deaminase(579 μM vs. 13.4 μM). This occurred despite the fact that the threoninedeaminase activity in the transformants containing overexpressedwild-type enzyme was much greater than that in transformants containingthe L481F mutant (4.20 u/mg vs. 0.59 u/mg in the absence of isoleucine).It is also noteworthy that the α-ketobutyrate concentrations reported inTable 3 are for a 1 ml extract solution from approximately 50 mg ofcells, and therefore represent a significant dilution of theintracellular metabolite, probably by a minimum factor of 10.

If simply increasing the levels of α-ketobutyrate was sufficient toproduce a significant 3HV component in the P(3HB-co-3HV) copolymer, thenthere should have been significantly more 3HV in the copolymer whenemploying pJM9238/pMON25683 than when employing pJM9238/pMON25660. Asshown in Table 3, this was not the case. This result suggests eitherthat the α-ketobutyrate was not being converted to propionyl-CoA, orthat the A. eutrophus PHB biosyntlietic enzymes encoded by pJM9238 couldnot efficiently catalyze formation of the C5 substrate((D)-3-hydroxy-valeryl-CoA, FIG. 2) for incorporation into the polymer.The results presented in Example 8 strongly suggest that this is atleast partially due to a block in the ability of the A. eutrophus PhbAto catalyze the condensation of acetyl-CoA and propionyl-CoA.

In summary, it is evident from the data in Tables 1-3 that mutant,deregulated threonine deaminases similar to those described hereinpossess a desirable deregulated phenotypic property useful in enhancingthe level of α-ketobutyrate in organisms such as bacteria and plantstargeted for the production of P(3HB-co-3HV). Isoleucine-deregulatedthreonine deaminases useful in the present invention preferably possessa level of isoleucine insensitivity such that at 100 μM isoleucine and10 mM threonine, the enzymes exhibit ≧20% activity relative to assayconditions in which isoleucine is absent. In addition to exhibitingreduced isoleucine sensitivity, threonine deaminases useful in thepresent invention can also exhibit reduced or no positive cooperativitycompared to the corresponding wild-type enzymes (Hill coefficient <1.5),and have kinetic parameters of K_(m) and V_(max) in the range of thewild-type enzyme (e. g., V_(max)=10-1000 U/mg, K_(m)=0.1 to 20 mM).

The present invention encompasses not only the DNA sequences shown inSEQ ID NOS: 5,7, and 8 and the proteins encoded thereby, but alsobiologically functional equivalent nucleotide and amino acid sequences.The phrase “biologically functional equivalent nucleotide sequences”denotes DNAs and RNAs, including chromosomal DNA, plasmid DNA, cDNA,synthetic DNA, and mRNA nucleotide sequences, that encode peptides,polypeptides, and proteins exhibiting the same or similar threoninedeaminase enzymatic activity as that of the mutant E. coli threoninedeaminases encoded by SEQ ID NOS: 5, 7, and 8, respectively, whenassayed enzymatically by the methods described herein, or bycomplementation. Such biologically functional equivalent nucleotidesequences can encode peptides, polypeptides, and proteins that contain aregion or moiety exhibiting sequence similarity to the correspondingregion or moiety of the E. coli IlvA muteins.

One can isolate threonine deaminases useful in the present inventionfrom various organisms based on homology or sequence identity. Althoughparticular embodiments of nucleotide sequences encoding deregulatedthreonine deaminases are shown in SEQ ID NOS: 5, 7, and 8, it should beunderstood that other biologically functional equivalent forms of suchderegulated threonine deaminase-encoding nucleic acids can be readilyisolated using conventional DNA-DNA or DNA-RNA hybridization techniques.Thus, the present invention also includes nucleotide sequences thathybridize to any of SEQ ID NOS: 5, 7, and 8 and their complementarysequences, and that code on expression for peptides, polypeptides, andproteins exhibiting the same or similar enzymatic activity as that ofthese deregulated threonine deaminases. Such nucleotide sequencespreferably hybridize to SEQ ID NOS: 5, 7, or 8 or their complementarysequences under moderate to high stringency (see Sambrook et al., 1989).Exemplary conditions include initial hybridization in 6× SSC, 5×Denhardt's solution, 100 mg/ml fish sperm DNA, 0.1% SDS, at 55° C. forsufficient time to permit hybridization (e.g., several hours toovernight), followed by washing two times for 15 min each in 2× SSC,0.1% SDS, at room temperature, and two times for 15 min each in 0.5-1×SSC, 0.1% SDS, at 55° C., followed by autoradiography. Typically, thenucleic acid molecule is capable of hybridizing when the hybridizationmixture is washed at least one time in 0.1× SSC at 55° C., preferably at60° C., and more preferably at 65° C.

The present invention also encompasses nucleotide sequences thathybridize under salt and temperature conditions equivalent to thosedescribed above to genomic DNA, plasmid DNA, cDNA, or synthetic DNAmolecules that encode the same amino acid sequences as these nucleotidesequences, and genetically degenerate forms thereof due to thedegenerancy of the genetic code, and that code on expression for apeptide, polypeptide, or protein that has the same or similar threoninedeaminase enzymatic activity as that of the deregulated threoninedeaminases disclosed herein.

Biologically functional equivalent nucleotide sequences of the presentinvention also include nucleotide sequences that encode conservativeamino acid changes within the amino acid sequences of the presentderegulated threonine deaminases, producing silent changes therein. Suchnucleotide sequences thus contain corresponding base substitutions basedupon the genetic code compared to the nucleotide sequences encoding thepresent deregulated threonine deaminases. Substitutes for an amino acidwithin the fundamental mutant deregulated E. coli threonine deaminaseamino acid sequences discussed herein can be selected from other membersof the class to which the naturally occurring amino acid belongs. Aminoacids can be divided into the following four groups: (1) acidic aminoacids; (2) basic amino acids; (3) neutral polar amino acids; and (4)neutral non-polar amino acids. Representative amino acids within thesevarious groups include, but are not limited to: (1) acidic (negativelycharged) amino acids such as aspartic acid and glutamic acid; (2) basic(positively charged) amino acids such as arginine, histidine, andlysine; (3) neutral polar amino acids such as glycine, serine,threonine, cyteine, cystine, tyrosine, asparagine, and glutamine; and(4) neutral nonpolar (hydrophobic) amino acids such as alanine, leucine,isoleucine, valine, proline, phenylalanine, tryptophan, and methionine.

Conservative amino acid changes within the present mutant deregulated E.coli threonine deaminase sequences can be made by substituting one aminoacid within one of these groups with another amino acid within the samegroup- While biologically functional equivalents of these mutantderegulated threonine deaminases can have any number of conservativeamino acid changes that do not significantly affect the threoninedeaminase enzymatic activity of this enzyme, 10 or fewer conservativeamino acid changes may be preferred. More preferably, seven or fewerconservative amino acid changes may be preferred; most preferably, fiveor fewer conservative amino acid changes may be preferred. The encodingnucleotide sequences (gene, plasmid DNA, cDNA, synthetic DNA, or mRNA)will thus have corresponding base substitutions, permitting them to codeon expression for the biologically functional equivalent forms of themutant deregulated E. coli threonine deaminases.

In addition to nucleotide sequences encoding conservative amino acidchanges within the amino acid sequences of the present mutant threoninedeaminases, biologically functional equivalent nucleotide sequences ofthe present invention also include genomic DNA, plasmid DNA, cDNA,synthetic DNA, and mRNA nucleotide sequences encoding non-conservativeamino acid substitutions, additions, or deletions. These include nucleicacids that contain the same inherent genetic information as thatcontained in the DNA of SEQ ID NOS:5, 7, and 8, and which encodepeptides, polypeptides, or proteins exhibiting the same or similarenzymatic activity as that of the present mutant deregulated threoninedeaminases. Such nucleotide sequences can encode fragments or variantsof these threonine deaminases. The enzymatic activity of such fragmentsand variants can be determined by complementation or enzymatic assays asdescribed above. These biologically functional equivalent nucleotidesequences can possess from 40% sequence identity, or from 60% sequenceidentity, or from 80% sequence identity, to 100% sequence identity tothe DNAs encoding the present deregulated threonine deaminases, orcorresponding regions or moieties thereof. However, regardless of thepercent sequence identity of these biologically functional equivalentnucleotide sequences, the encoded proteins would possess the same orsimilar enzymatic activity as the deregulated threonine deaminasesdisclosed herein. Thus, biologically functional equivalent nucleotidesequences encompassed by the present invention include sequences havingless than 40% sequence identity to any of SEQ ID NOS: 5, 7, and 8, solong as they encode peptides, polypeptides, or proteins having the sameor similar enzymatic activity as the deregulated threonine deaminasesdisclosed herein.

Mutations made in E. coli threonine deaminase cDNA, chromosomal DNA,plasmid DNA, synthetic DNA, mRNA, or other nucleic acid preferablypreserve the reading frame of the coding sequence. Furthermore, thesemutations preferably do not create complementary regions that couldhybridize to produce secondary mRNA structures, such as loops orhairpins, that would adversely affect mRNA translation.

Useful biologically functional equivalent forms of the DNAs of SEQ IDNOS:5, 7, and 8 include DNAs comprising nucleotide sequences thatexhibit a level of sequence identity to corresponding regions ormoieties of these DNA sequences from 40% sequence identity, or from 60%sequence identity, or from 80% sequence identity, to 100% sequenceidentity to the DNAs encoding the present deregulated threoninedeaminases, or corresponding regions or moieties thereof. However,regardless of the percent sequence identity of these nucleotidesequences, the encoded proteins would possess the same or similarenzymatic activity as the deregulated threonine deaminases disclosedherein. Thus, biologically functional equivalent nucleotide sequencesencompassed by the present invention include sequences having less than40% sequence identity to any of SEQ ID NOS:5, 7, and 8, so long as theyencode peptides, polypeptides, or proteins having the same or similarenzymatic activity as the deregulated threonine deaminases disclosedherein. Sequence identity can be determined using the “BestFit” or “Gap”programs of the Sequence Analysis Software Package, Genetics ComputerGroup, Inc., University of Wisconsin Biotechnology Center, Madison, Wis.53711.

Due to the degeneracy of the genetic code, i.e., the existence of morethan one codon for most of the amino acids naturally occuring inproteins, genetically degenerate DNA (and RNA) sequences that containthe same essential genetic information as the DNAs of SEQ ID NOS:5, 7,and 8 of the present invention, and which encode the same amino acidsequences as these nucleotide sequences, are encompassed by the presentinvention. Genetically degenerate forms of any of the other nucleic acidsequences discussed herein are encompassed by the present invention aswell.

The nucleotide sequences described above are considered to possess abiological function substantially equivalent to that of the mutant E.coli threonine deaminase-encoding DNAs of the present invention if theyencode peptides, polypeptides, or proteins having isoleucine-deregulatedthreonine deaminase enzymatic activity differing from that of any of themutant E. coli threonine deaminases disclosed herein by about +30% orless, preferably by about ±20% or less, and more preferably by about±10% or less when assayed in vivo by complementation or by the enzymaticassays discussed above.

EXAMPLE 3 Increased Production of α-Ketobutyrate Via Use of theThreonine Deaminase of Rhodospirillum rubrum

The phototrophic, non-sulfur purple bacterium Rhodospirillum rubrum(ATCC 25903) is the source of a natural deregulated threonine deaminase(Feldberg and Datta, 1971). As shown in Tables 1 and 2, above, thethreonine deaminase from R. rubrum displays normal Michaelis-Mentenbehavior, and is not significantly feedback inhibited by isoleucine.This enzyme is also unaffected by activators of the degradative enzyme(Feldberg and Datta, 1971). The R. rubrum threonine deaminase cantherefore also be expressed in bacteria and plants in order to enhancethe production of α-ketobutyrate from L-threonine.

SDS-PAGE analysis indicates that the monomeric molecular weight of theR. rubrum threonine deaminase is approximately 42 kD (data not shown).This is intermediate between the molecular weight of the E. colibiosynthetic (56 kD) and biodegradative enzymes (35 kD). The R. rubrumenzyme may therefore represent a third class of threonine deaminaseuseful in the present invention. Möckel et al. (1994) similarly observedthat the threonine deaminase of C. glutamicium was of intermediatemolecular weight, having a C-terminal deletion of 95 amino acids.

From what is known of other threonine deaminases, it is probable thatthe C-terminal domain of the R. rubrum enzyme is truncated, resulting inthe deregulated phenotype of the enzyme. Taillon et al. (1988) publishedan amino acid comparison of three biosynthetic threonine deaminases andone biodegradative threonine deaminase. This comparison revealedN-terminal conservation between the two forms of threonine deaminase.The N-terminus appears to be involved in catalysis. The biodegradativeform of the enzyme is truncated at the C-terminus, and it has beensuggested that this deletion results in the isoleucine-deregulatedphenotype (Taillon et al., 1988). Based on SDS-PAGE analysis of the R.rubrum threonine deaminase which indicates that the R. rubrum enzymeposses a molecular weight intermediate between that of the biosyntheticand degradative enzymes, the R. rubrum threonine deaminase may have atruncated C-terminus, effectively deleting the regulatory domain.

EXAMPLE 4 Increased Production of α-Ketobutyrate Using Other Modified orNaturally-Occurring Forms of Threonine Deaminase

Deregulated threonine deaminases useful in the present invention can beeither naturally-occurring, or produced by recombinant DNA techniquessuch as those employed in Example 2. If naturally-occurring, suchthreonine deaminases can be identified by screening organisms forthreonine deaminase activity, followed by measuring the sensitivity ofthat activity to isoleucine inhibition using the standard assay methodsdescribed above. Genes for these enzymes can then be isolated bycomplementation into threonine deaminase-negative bacteria (Fisher andEisenstein, 1993). The levels of isoleucine analogs such aL-O-methylthreonine (OMT) in the growth medium can be varied during thecomplementation procedure to select organisms that expressisoleucine-deregulated biosynthetic threonine deaminases. Alternatively,genes for threonine deaminases can be obtained by hybridizationtechniques using an appropriate probe, preferably based upon thenucleotide sequence comprising the N-terminal region of the biosyntheticthreonine deaminase. Following expression of these isolated genes in anappropriate host, they can be tested for isoleucine insensitivity asdescribed previously.

Alternatively, deregulated threonine deaminases can be created byphysical or chemical mutagenesis of wild-type threonine deaminase genesutilizing a variety of techniques known in the art to introduce eitherspecific or random mutations into such genes (Eisenstadt et al., 1994;as described in Example 2; Feldmann et al., 1994). Muteins created inthis way can be analyzed by methods such as those described in Example 2to determine catalytic efficiencies and the extent of inhibition byisoleucine.

One strategy for creating deregulated threonine deaminases useful in thepresent invention is based upon knowledge of the functional domains ofthis enzyme. The functional domains of both the degradative andbiosynthetic types of threonine deaminase have been analyzed (Taillon etal., 1988). All known mutations affecting feedback inhibition of thebiosynthetic threonine deaminase are C-terminal region mutations. If thebiosynthetic threonine deaminase is compared to the biodegradativethreonine deaminase at the amino acid level, it can be seen that theregions of homology between the two forms exist only in the N-terminalregions of the polypeptides. Since the biodegradative form of the enzymeis not regulated by isoleucine, the N-terminal region is likelyresponsible for the catalytic activity, while the C-terminal region islikely involved in the feedback regulation (Fisher and Eisenstein,1993). This knowledge of the regulatory domains of the enzyme suggeststhat modification of the C-terminal region will produce mutants withdesirable properties for the purposes disclosed herein. For example,portions of the C-terminal region of the biosynthetic threoninedeaminase can be sequentially deleted, and the remainder of the enzymepolypeptide assayed for activity to isolate deregulated deletionmutants. Deletions of the C-terminal region of the biosyntheticdeaminase can be produced by a variety of techniques known in the art.For example, Exonuclease III can be used to create a ladder of 3′deletions. Following expression of the truncated genes, the enzymaticactivity of the modified deaminases can be assayed as described above.

Alternatively, various domains of different threonine deaminases can bereplaced with one another to determine their effect on enzymeregulation. For example, the C-terminal region of a biosyntheticthreonine deaminase can be replaced with the C-terminal region of abiodegradative threonine deaminase such as that from E. coli, and theresulting polypeptide assayed for feedback inhibition by isoleucine.Domain swapping has been discused in Cohen and Curran 1990; Czerny et.al., 1993; and Zinszner et. al., 1994.

Mutants of biodegradative threonine deaminase that are not inhibited bypyruvate, or which do not require activation by AMP, can be generated byrandom mutagenesis of a cloned biodegradative threonine deaminase, or byswapping domains of the biodegradative threonine deaminase with those ofthe biosynthetic enzyme. Such mutant biodegradative threonine deaminasescan be use to generate high levels of α-ketobutyrate in plants orbacteria. No biodegradative threonine deaminase has yet been reportedthat does not require a metabolite for activation.

EXAMPLE 5 Expression and Activity of Wild-type and Mutant ThreonineDeaminases in Plant Cells Expression and Activity in ElectroporatedTobacco Protoplasts

In order to test the expression and enzymatic activity of the cloned E.coli biosynthetic threonine deaminases in plant cells, several plantexpression plasmids were constructed for transient expression in tobaccoleaf protoplasts. The plant expression plasmids contain all thenecessary elements for plant cell expression, including a promoter(i.e., e35S (Odell et al., 1985)), the 5′ untrans-lated leader sequencewithin the e35S promoter, a coding sequence, and a transcriptiontermination and polyadenylation signal (i.e., E9 (Coruzzi et al.,1984)). The wild-type E. coli biosynthetic threonine deaminase (IlvA)encoded by the insert in pMON25663 (FIG. 13) and the three mutant,deregulated E. coli biosynthetic threonine deaminases encoded by theinserts in pMON25686 (FIG. 14), pMON25687 (FIG. 15), and pMON25688 (FIG.16) containing the IlvA 219 (L447F), IlvA466 (L481F), and IlvA219/466(L447F/L481F) mutations, respectively, were cloned downstream of thee35S constitutive plant promoter (Odell et al., 1985) and targeted tochloroplasts using a translational fusion to the Arabidopsis rubiscosmall subunit chloroplast transit peptide (ArabSSU1A; Stark et al.,1992). The four plasmids containing the various threoninedeaminase-encoding DNAs were independently electroporated into tobaccoleaf protoplasts by the method of Hinchee et al. (1994). Tobaccoprotoplasts electroporated with the various DNA's were sonicated to lysethe protoplasts. FIG. 17 shows the expression of the various IlvAenzymes determined by Western blot analysis using rabbit polyclonalantibodies generated to the wild-type IlvA. Enzyme activity wasmonitored in extracts of lysed protoplasts in the presence and absenceof isoleucine as described above, and is reported in Table 4.

TABLE 4 Acvtivity of Threonine Deaminases Introduced Into TobaccoProtoplasts Threonine Deaminase Specific Activity, u/mg (%)¹Electroporated Plasmid 0 mM Isoleucine 1 mM Isoleucine no plasmid DNA0.000 0.000 pMON25663 (wt IlvA) 0.089 0.006 (6.7) pMON25686 0.101 0.091(90.1) (IlvA 219 L447F) pMON25687 0.162 0.048 (29.6) (IlvA 466 L481F)pMON25688 0.181 0.181 (100) (IlvA 219/466 L447F/L481F) ¹Rates weredetermined using 10 mM threonine as substrate, at the indicatedconcentrations of isoleucine. Values in parenthesis are % activityrelative to the results with 0 mM isoleucine.

As shown in FIG. 17, the “no DNA” control tobacco protoplasts did notexhibit any immunoreactive bands corresponding to threonine deaminase byWestern blotting. In contrast, extracts of lysed tobacco protoplastsinto which plasmids pMON25663, pMON25686, pMON25687, and pMON25688 wereelectroporated contained immunoreactive bands corresponding to thepredicted molecular weight of the threonine deaminase encoded by theinserts of the respective plasmids. The enzymatic activity data reportedin Table 4 demonstrate that both wild-type and deregulated threoninedeaminases exhibit their predicted isoleucine deregulated enzymaticactivities when expressed in plant cells based upon the results reportedin the section entitled “Biochemical Analysis of Wild-type and Mutant E.coli Threonine Deaminases” of Example 2.

Expression and Activity in Transformed Soybean Callus

As shown above, the E. coli IlvA proteins can be functionally expressedin a plant transient expression system. In order to test the expressionof a biosynthetic threonine deaminase in a stably transformed planttissue, non-differentiating callus derived from soybean hypocotyl tissuewas transformed with a plasmid containing the E. coli wild-type ilvAthreonine deaminase gene using Agrobacterium tumefaciens ABI (Koncz andSchell, 1986).

Transformation Vector

Soybean callus tissue was transformed with binary Ti plasmid pMON25668(FIG. 18). Plasmid pMON25668 contains the e35S-expressed,plastid-targeted (ARABSSU1A) E. coli wild-type ilvA threonine deaminasegene, the e35S-expressed, plastid-targeted (ARABSSU1A) A. eutrophus phbA(β-ketothiolase) gene, and the e35s-expressed neomycinphosphotransferase type II (NptII) gene for kanamycin resistance.pMON26668 contains two border sequences for T-DNA transfer into theplant chromosome. The right border sequence is the only border requiredfor T-DNA transfer into the plant; however, including the left bordersequence terminates T-DNA integration at that point, thereby limitingthe unnecessary incorporation of “extra” DNA sequences into the plantchromosome. pMON25668 also contains the minimun sequence of ori-322 forreplication and maintence in E. coli as well as ori-V of the broad hostorigin RK2 for replication in Agrobacterium. TrfA is supplied in transfor proper replication in Agrobacterium. Further details of thesevectors can be found in Glick et al., (1993) and the references citedtherein.

Seedling Growth

The bottom of a 100×25 mm Petri dish was covered with Asgrow A3237soybean seeds and the lid was replaced. Four Petri dishes containingseeds were placed inside a vacuum bubble containing a glass beakerholding 200 mls of Chlorox (sodium hypochlorite). The bubble was placedin a chemical fume hood. Two mls of HCl were added to the Chlorox, andthe bubble lid was placed on quickly. A vacuum was pulled just longenough for the lid to stay on tightly, and the seeds were exposed to theresulting fumes overnight. The following day, the Petri dishes wereremoved from the bubble, and dry Captan (⅛ tsp) was added to each dish.The seeds were covered with sterile water and incubated in the Captanslurry for 5-7 minutes, after which the Captan slurry was removed.

After pipetting off the liquid from all of the plates, the seeds (20-25per plate) were placed on 0.8% water agar plates. Only good qualityseeds were chosen, i.e., no seeds with cracked seedcoats, discoloration,or other damage were used. Five plate high stacks of Petri plates werethen wrapped with a rubberband, covered with a clear plastic bag, andincubated in a warm room set at 25° C. under continuous cool white light(60 μEn m⁻²s⁻¹; this can range from about 20-80 μEn m⁻²s⁻¹) for 6 daysto obtain seedlings.

Preparation of Agrobacterium

About one week prior to inoculation, Agrobacterium tumefaciens ABI wasstreaked from a glycerol stock onto LB agar-solidified plates (1.5%agar) containing 100 mg/; spectinomycin, 50 mg/l kanamycin, and 25 mg/lchloramphenicol, and grown at room temperature.

The day before explant inoculation, Agrobacterium cultures were startedin clear plastic tubes containing 2 mls of YEP (Sambrook et al., 1989)containing the same levels of spectinomycin, kanamycin, andchloramphenicol as above. About ¼ loopful of bacteria was placed in eachYEP tube. The tubes were placed on a rotator in a warm room set at 25°C. Eight hours later, each 2 ml culture was added to 25 mls liquid YEPcontaining the same amounts of antibiotics as above, as well as 200 μMacetosyringone and 1 mM galacturonic acid, in a sterile 250 ml flask.The cultures were grown overnight in the dark at 28° C. with shaking(170 rpm).

On the day of explant inoculation, 12 ml aliquots of Agrobacterium wereplaced in sterile 50 ml centrifuge tubes. The tubes were centrifuged for12 minutes at 2,000 rpm in order to pellet the cells. The supernatantswere poured off and the pellets were resuspended in 20 mls of co-culturemedium containing {fraction (1/10)} MS salts and {fraction (1/10)} B5vitamins (Sigma, M 0404) and 15 g/l glucose, 20 mM MES, pH 5.4, andcombined. The cell density was adjusted to a final OD₆₆₀ in the rangefrom 0.3-0.35 from an initial OD₆₆₀ of 0.5-0.6. Thirty mls of thisAgrobacterium suspension were used to inoculate each batch of 50hypocotyl explants.

Explant Inoculation and Co-culturing

One day prior to explant inoculation, the plates containing the 6 dayold seedlings were taken from the warm room and placed in a refrigeratorat 0-1 0° C. (average temperature of 4° C.) for approximately 24 hrs. Onthe day of explant inoculation, the stacks of seedlings were taken fromthe cold one at a time just prior to explanting to maintain coldness.The hypocotyls were cut into 5 mm sections, and batches of 50 sectionswere inoculated by incubating them with 30 mls of Agrobacteriumsuspension in a Petri plate (100×25 mm) for 30 min. After 30 min, theAgrobacterium suspension was pipetted off. The explants were blotted onsterile qualitative Whatman filter paper, and then placed 10 per platein 100×15 mm Petri plates containing one 8.5 cm sheet of sterile Whatmanfilter paper and 4 mls/plate of liquid co-culture medium containing{fraction (1/10)} MS salts and {fraction (1/10)} B5 vitamins, 15 g/lglucose, 3.9 g/l MES, 4.68 mg/l naphthaleneacetic acid (this can rangefrom about 1-8 mg/l), 2.5 mg/l kinetin (this can range from about 0.5-4mg/l), 200 μM acetosryingone (this can range from about 50-300 μM), and1 mM galacturonic acid (this can range from about 0.1-2 mM), at pH 5.4.The plates were wrapped with parafilm and incubated for two days in awarm room set at 25° C. under continuous cool white light (40 μEnm⁻²s⁻¹; this can range from about 20-60 μEn m⁻²s⁻¹).

Selection

After the two day co-culture period, the explants were placed 10 perplate onto solid selection medium containing 1× MS salts and 1× B5vitamins (Sigma, M 0404), 3% sucrose (this can range from about 1-6%),4.68 mg/l naphthalene-acetic acid (this can range from about 1-8 mg/l),2.15 mg/l kinetin (this can range from about 0.5-4 mg/1), 500 mg/lticarcillin (this can range from about 25-250 mg/l), 100 mg/l kanamycin(this can range from about 25-250 mg/l), and 0.7% purified agar (Sigma,B11853), at pH 5.7. The plates were wrapped with white 3M filter tapeand cultured in a warm room set at 25° C. under continuous cool whitelight (40 μEn m⁻²s⁻¹; this can range from about 20-60 μEn m⁻²s⁻¹).Explants were transferred to fresh medium every two weeks. At the sixweek timepoint, the calli were excised from the hypocotyls and againtransferred to the same selection medium for an additional two weeks.All calli were maintained on this selection medium until they were readyfor assaying at 10-16 weeks.

Kanamycin-resistant calli were analyzed by Western blotting andenzymatic assays as described above to determine if E. coli IlvA wasexpressed and exhibited enzymatic activity in stably transformed soybeancells. The results are shown in FIG. 19.

The data in FIG. 19 demonstrate that in four transformed calli (i.e.,518-4, 518-15, 518-16, and 518-17), the E. coli IlvA biosyntheticthreonine deaminase was detectable by Western blot analysis. Enzymaticactivity of the IlvA was also confirmed in the same transformed calli,correlating with the positive Western events. These results demonstratethat the E. coli IlvA can be expressed and maintain enzymatic activityin stably transformed plant tissue.

Colau et al., (1987) expressed the S. cerevisiae wild-type Ilv1 gene intobacco (N. plumbaginifolia) mutants deficient in threonine deaminase.Expression of this threonine deaminase in these mutants was sufficientto complement the isoleucine auxotrophy.

EXAMPLE 6 Increased Propionyl-CoA Production From α-Ketobutyrate ViaModification of the Pyruvate Dehydrogenase Complex or Expression of aBranched-Chain α-Ketoacid Dehydrognase Complex

As demonstrated for the bacterial (Danchin et al., 1984; Bisswanger,1981) and pea chloroplast complexes (Camp et al., 1988; Camp andRandall, 1985), pyruvate dehydrogenase complex (PDC) catalyzes theoxidative decarboxylation of α-ketobutyrate to produce propionyl-CoA(FIG. 3). The rates of turnover of α-ketobutyrate with both thebacterial and chloroplast PDCs are approximately 10% of that relative topyruvate at 1.5 mM concentration of α-ketoacid. The effect on kineticconstants with the bacterial enzyme is a 10-fold increase in K_(m), anda five-fold decrease in V_(max) (Bisswanger, 1981). Therefore, ifsteady-state concentrations of α-ketobutyrate rise to high μM or low mMlevels, turnover of α-ketobutyrate to propionyl-CoA catalyzed by thepyruvate dehydrogenase complex should occur in plants and bacteria. VanDyk and LaRossa (1987) demonstrated in wild-type Salmonella typhimurium,under conditions where acetohydroxyacid synthase (AHAS, FIG. 3) activityis reduced by inhibition with sulfometuron methyl, that theconcentration of α-ketobutyrate increases significantly, andpropionyl-CoA accumulates (acetohydroxyacid synthase condensesα-ketobutyrate and pyruvate to produce 2-aceto-2-hydroxy-isovalerate asthe second step in the biosynthesis of isoleucine, see FIG. 3). Withrespect to the P(3HB-co-3HV) copolymer biosynthetic pathway depicted inFIG. 3 and as discussed in Examples 2-4 above, high levels ofα-ketobutyrate produced by the overexpression of a deregulated threoninedeaminase should be sufficient for propionyl-CoA formation, even whenthere is competing demand for the α-ketoacid in isoleucine biosynthesis.Results presented in Table 3 demonstrating elevated levels ofα-ketobutyrate in vivo from the presence of a deregulated threoninedeaminase support this hypothesis.

The pyruvate dehydrogenase complex is a multienzyme complex thatcontains three activities: a pyruvate decarboxylase (E1), adihydrolipoyl transacetylase (E2), and a dihydrolipoyl dehydrogenase(E3). Other α-ketoacid dehydrogenase complexes exist that use the samecatalytic scheme with α-ketoacids other than pyruvate. The TCA cycleα-ketoglutarate dehydrogenase complex is an example. The branched-chainα-ketoacid dehydrogenases are also multienzyme complexes constructed ina manner similar to that of the pyruvate dehydrogenase complex. If theendogenous pyruvate dehydrogenase complex is not sufficiently active incatalyzing the turnover of α-ketobutyrate to propionyl-CoA for copolymerproduction, it can be modified to enhance its activity.

One way to accomplish this, for example, is to overexpress abranched-chain α-ketoacid decarboxylase E1 subunit having better bindingand decarboxylating properties with α-ketobutyrate than does the nativePDC E1. For example, the branched chain α-ketoacid dehydrogenase complexof bovine kidney has substantial activity with α-ketobutyrate assubstrate, comparable to that with the normal substrateα-ketoisovalerate (K_(m)=56 μM and 40 μM, respectively; specificactivity=12†μmol/min•mg, Pettit et al., 1978). Other examples ofbranched chain α-ketoacid dehydrogenase compleses are those fromPseudomonas putida (Burns et al, 1988), and Bacillus subtilis (Lowe etal, 1983). An overexpressed branched-chain α-ketoacid decarboxylase E1subunit could effectively compete with the endogenous pyruvatedehydrogenase complex E1 subunit, and combine with the pyruvatedehydrogenase complex E2E3 subcomplex to create a functional hybridcomplex. This has in fact been shown to occur naturally with the E. coliα-ketoglutarate dehydrogenase complex, where approximately 10% of thetotal E1 component is the pyruvate dehydrogenase complex E1 (Steginskyet al., 1985). The pyruvate dehydrogenase complex E1 component accountsfor the pyruvate activity of the α-ketoglutarate dehydrogenase complex.An artificially produced hybrid of E1 (pyruvate decarboxylase) with E2E3(dihydrolipoyl transsuccinylase-dihydrolipoyl dehydrogenase subcomplex)has higher turnover rates with pyruvate compared to the naturallyisolated aketoglutarate dehydrogenase complex, but understandably lowerrates than that of the pyruvate dehydrogenase complex itself (Steginskyet al., 1985).

Alternatively, both the E1 and E2 components of a branched-chainα-ketoacid dehydrogenase complex that has significant activity withα-keto-butyrate, such as the bovine kidney complex mentioned above, canbe expressed. Since the dihydrolipoyl dehydrogenase (E3) is common toall α-ketoacid dehydrogenase complexes, and would be naturally producedfor the pyruvate dehydrogenase complex, overexpression of this componentmay not be necessary. The endproduct would be a filly functional,branched-chain α-keto-acid dehydrogenase complex. If additional E3 isrequired to interact with overexpressed and endogenous E1 and E2components, one could overexpress a plant or bacterial E3 (Camp &Randall, 1985, and Camp et al., 1988), or the specific E3 of thebranched-chain α-ketoacid dehydrogenase complex, for example the bovinekidney complex (Pettit et al., 1978).

In addition to an E1 α-ketoacid decarboxylase catalyzing thedecarboxylation of an α-ketoacid in an intact complex, unassociated E1in the presence of an oxidizing agent will catalyze the decarboxylationof an α-ketoacid to form CO₂ and the free acid (Gruys et al., 1989;Speckhard & Frey, 1975; Reed & Willms, 1966). Thus, it is possible thatan unassociated branched-chain E1, such as that from bovine kidney, whenoverexpressed in plants or bacteria, will produce propionate and CO₂from α-ketobutyrate in the presence of an endogenous oxidizing agentsuch as ferredoxin. Propionate can be activated to propionyl-CoA byendogenous or overexpressed acyl-CoA synthetase (refer to Example 7).

Mutagenesis of the endogenous E1 of the host PDC is another approach toenhancing its activity toward α-ketobutyrate. Recent reports ofmutagenesis of the E2 component from Bacillus (Wallis & Perham, 1994),Azotobacter (Schulze et al., 1992 & 1993), and the human E3 component(Kim and Patel, 1992), have demonstrated that one can modify both theactivity of these enzymes towards substrates as well as their bindingaffinity to the complex. Similar manipulations can be carried out on theE1 component to enhance its catalytic turnover of α-ketobutyrate.

EXAMPLE 7 Increased Propionyl-CoA Production From α-Ketobutyrate ViaOverexpression of Pyruvate Oxidase and ACI-CoA Synthetase

Another method of enhancing the level of propionyl-CoA in bacteria andplants involves, in a first step, the oxidative decarboxylation ofα-ketobutyrate (the amount of which can be increased in vivo byutilizing a deregulated threonine deaminase as described above inExamples 2-4), to propionate and CO₂, catalyzed by pyruvate oxidase(E.C. 1.2.3.3) (FIG. 3). Unpublished results have shown that pyruvateoxidase efficiently catalyzes this reaction (J. E. Cronan, University ofIllinois, personal conmmunication). The poxB gene of E. coli encodes thepyruvate oxidase enzyme (Grabau and Cronan, 1986). Another example isthe pyruvate oxidase from Lactobacillus plantarum (Miller and Schulz1993). This homotetrameric protein of 62 kDa has been thoroughlycharacterized (Chang & Cronan, 1995, and references cited therein).

In a second step following pyruvate oxidase, endogenous bacterial orplastid acyl-CoA synthetases such as acetyl-CoA synthetase will activatethe free propionate to propionyl-CoA (Doi et al., 1986; Nakamura et al.,1991). Yeast acetyl-CoA synthetase has been shown to catalyze theactivation of propionate to propionyl-CoA (Patel and Walt, 1987). Inaddition to yeast, the A. eutrophus acoE gene has been cloned and shownto be active with propionyl-CoA (Priefert and Steinbüchel, 1992). If theendogenous acetyl-CoA synthetase activity proves to be insufficient forproducing propionyl-CoA, the enzyme, for example that from yeast, can beoverexpressed in the plant, or in a bacterial host.

Another system for activating propionate to propionyl-CoA has beendescribed by Rhie and Dennis (1995). In E. coli, the actions of acetatekinase (acke) and phosphotransacetylase (pta) are predominatelyresponsible for the conversion of propionate to propionyl-CoA. Thusacetyl-CoA synthetase can be replaced by acetate kinase (acke) andphosphotransacetylase (pta) in the present invention.

Enhanced propionyl-CoA production can thus be achieved by overexpressingany of the foregoing enzymes in a plant or bacterial host.Overexpression of the foregoing enzymes in combination with anoverexpressed deregulated threonine deaminase and overexpressed PHAβ-ketothiolase, β-ketoacyl-CoA reductase, and PHA synthase enzymes isexpected to result in the production of P(3HB-co-3HV) copolymer.

EXAMPLE 8 Optimization of β-Ketothiolase, β-Ketoacyl-CoA Reductase, andPHA Synthase Activity for Production of P(3HB-co-3HV) Copolymer inBacteria and Plants

Alcaligenes eutrophus can produce both PHB homopolymer and, whenprovided with an appropriate precursor such as propionate, P(3HB-co-3HV)copolymer (U.S. Pat. No. 4,477,654). It was expected that P(3HB-co-3HV)copolymer would also be produced if E. coli transformed with the A.eutrophus PHB biosynthetic genes was provided with the appropriateprecursor, for example, propionate or α-ketobutyrate (see FIG. 3).

The experiment reported in Table 3, supra, was designed in part to testthis hypothesis. Introduction of a deregulated threonine deaminase(L481F) into E. coli was expected to provide α-ketobutyrate forincorporation of a C5 monomer into the PHA polymer. However, the data inTable 3 demonstrate that while the α-ketobutyrate concentration in cellscontaining the overexpressed threonine deaminase was dramaticallyelevated, PHA polymer content and composition were essentially identicalin both wild-type cells and cells expressing the introduced threoninedeaminase. These results suggest the presence of a metabolic block inthe conversion of α-ketobutyrate to P(3HB-co-3HV) copolymer.

As shown in FIG. 3, there are four reactions required to convertα-ketobutyrate to P(3HB-co-3HV) copolymer. These reactions are catalyzedsuccessively by the pyruvate dehydrogenase complex, β-ketothiolase,acetoacetyl-CoA reductase, and PHB synthase. The metabolic block maytherefore involve the inability of one of these enzymes to utilizesubstrate derived from α-ketobutyrate. However, the literature suggeststhat all of these enzymes possess the appropriate substratespecificities (Bisswanger, 1981; Haywood et al., 1988a; Haywood et al,1988b; Haywood et al, 1989). Thus, the substrate specificities of someof these enzymes were reevaluated.

Substrate Specificity of PhbB Acetoacetyl-CoA Reductase from A.eutrophus and β-Ketothiolases from Various Sources

PhbB Acetoacetyl-CoA Reductase

Analysis of A. eutrophus PhbB acetoacetyl-CoA reductase substratespecificity in the forward reaction was carried out usingβ-ketobutyryl-CoA (acetoacetyl-CoA) and β-ketovaleryl-CoA. Theseβ-ketoacyl-CoAs were reduced to the corresponding D-β-hydroxyacyl-CoAsusing NADPH (FIG. 1) This analysis was previously performed by Haywoodet al. (1988a), but in the reverse reaction where in situ-generatedD-β-hydroxyacyl-CoA was oxidized with NADP⁺. It is important toestablish that this enzyme can catalyze the forward reaction with betβ-ketovaleryl-CoA and NADPH to be confident that it does not represent ametabolic block in copolymer production.

E. coli transformed with plasmid pMON25628 (FIG. 20) encoding A.eutrophus PhbB acetoacetyl-CoA reductase were grown on LB to early logphase and induced with nalidixic acid (60 μg/ml). Protein extracts wereobtained by pelleting induced cells, resuspending in 50 mM KPi and 5%glycerol, sonicating, and removing cellular debris by centrifugation.The assay was conducted at pH 7.0 with 100 mM KPi, 0.15 mM NADPH, and 60μM β-ketoacyl-CoA. The reaction was initiated with acetoacetyl-CoAreductase. Synthesis of β-ketovaleryl-CoA was accomplished through ascaled-up version (10 μmol of acyl-CoA in 2 ml total volume) of the insitu-generated β-ketoacyl-CoA procedure described below in the sectionentitlted “Thiolysis Activity of A. eutrophus BktB.” Purification wasaccomplished through semi-prep C8-reverse-phase HPLC using the followinggradient per sample run (Buffer A is 100 mM ammonium acetate, pH 6.0, Bis acetonitrile) with a 4.0 ml/min flow rate: 0-25 min, 5-45% B; 25-30min, hold at 45% B; 30-32 min, 45-5% B; 32-42 min, hold at 5% B.β-ketovaleryl-CoA eluted and was collected from 8.5 to 10.5 mm.

Rate results for β-ketobutyryl-CoA and β-ketovaleryl-CoA with PhbBacetoacetyl-CoA reductase gave specific activities of 103 and 28units/mg protein, respectively, which is 27% relative rate for the C5versus the C4 substrate under the conditions of the assay. This isconsistent with the results described by Haywood et al. (1988a), butfurther demonstrates that this enzyme is catalytically sufficient forthe metabolic conversion of β-ketovaleryl-CoA to D-β-hydroxyvaleryl-CoAin the production of copolymer.

β-Ketothiolases

Condensation Activity of A. eutrophus PhbA and BktB

The data presented above demonstrate that the substrate specificity ofPhbB is sufficient for production of C5 monomer for incorporation intoPHA. Therefore, the substrate specificity of PhbA, as well as of otherA. eutrophus β-ketothiolases, was investigated.

As noted above, A. eutrophus can produce P(3HB-co-3HV) copolymer whenprovided with an appropriate C5 precursor. Slater et al. (1988)demonstrated the presence of genes other than phbA encodingβ-ketothiolase activity in A. eutrophus. One of these β-ketothiolases,designated BktB herein, was encoded on plasmid pBK6. A. eutrophus BktBwas obtained from E. coli DH5α transformed with plasmid pMON25754.pMON25754 was produced from the A. eutrophus pBK6 clone (Slater et al.,1988) by deleting a 5.0 kb XhoI fragment (there is an additional XhoIsite approximately 5.0 kb 5′ to the XhoI site shown in FIG. 1 of Slateret al.). The pBK6 clone was produced by Slater et al. (1988) from A.eutrophus H16, i.e., ATCC accession number 17699, which is publiclyavailable from the American Type Culture Collection, 12301 ParklawnDrive, Rockville, Md. 20852. BktB was overexpressed from its nativepromoter on the high copy plasmid pMON25754 by growing the cellsovernight at 37° C. to stationary phase in LB broth containingampicillin (100 μg/ml). A. eutrophus PhbA was obtained from E. coli DH5αtransformed with pMON25636 (FIG. 22) by growing the cells at 37° C. inLB broth containing ampicillin (100 μg/ml) to early log phase and theninducing with IPTG. Cells were harvested after two hours of furthergrowth. PhbA and BktB protein extracts were prepared by pelleting thecells, resuspending in 50 mM KPi and 5% glycerol, sonicating, andremoving cellular debris by centrifugation.

The activity of β-ketothiolases can be assayed in both the condensationand thiolysis directions. However, condensation activity for productsthat are the result of a mixed condensation of acetyl-CoA and a longerchain acyl-CoA are difficult to measure. This is due to the difficultyin distinguishing between acetoacetyl-CoA, the product of condensing twoacetyl-CoA molecules, and the mixed condensation reaction product, bothof which form simultaneously. To date, condensation assays have beenspectrophotometrically-based, but these are valid only when there is asingle acyl-CoA substrate present (i.e., acetyl-CoA) since they cannotdiscriminate between the products formed. The assay described belowrepresents the first reported assay that permits complete quantitationof all condensation products regardless of the nature of startingsubstrates.

β-ketothiolase assays designed to measure the condensation activity withacetyl-CoA plus either propionyl-CoA or butyryl-CoA were performed using1-¹⁴C-labelled acetyl-CoA. Since the equilibrium for the reaction liesheavily toward the starting acyl-CoAs rather than the condensedβ-ketoacyl-CoA product, the latter was “pulled” to the correspondingβ-hydroxyacyl-CoA using NADH and β-ketoacyl-CoA dehydrogenase. Inaddition, to avoid the strong feedback inhibition of β-ketothiolase byfree CoA, each assay solution contained 5,5′-dithio-bis(2-nitrobenzoicacid) (DTNB) to trap chemically the free CoA produced by the reaction.

The condensation assay mixture (0.5 ml final volume) contained 100 mMKPi, pH 7.8, 0.5 mM NADH, 0.30 mM 1-¹⁴C-acetyl-CoA (0.15 μCi), 5 unitsβ-ketoacyl-CoA dehydrogenase, 0.5 mM DTNB, and variable propionyl- orbutyryl-CoA as indicated in Table 5. The reaction was initiated by theaddition of β-ketothiolase. Following 20 minutes at 30° C., eachreaction mixture was quenched by the addition of 25 μl of 10% formicacid. Thirty μl of 25% H₂SO₄ were added to 200 μl of quenched reactionmix; this mixture was then incubated at 70° C. for 24 hours to hydrolyzeall CoA thioesters to their free acids. Sixty μl of 5 M sodium formatewere added to the sample to neutralize the pH, followed by analysis of a130 μl aliquot using C18-reverse-phase HPLC (0.5×25 cm column).Detection and quantitation were carried out by monitoring flow-throughwith a Radiomatic flow detector. A gradient elution was employed usingaqueous 1% acetic acid (solvent A) and acetonitrile (solvent B). Thefollowing gradient program was used per sample run with a flow rate of1.0 ml/min: 0-15 min, 0-35% B; 15-20 min hold at 35% B; 20-21 min, 35-0%B; 21-30 min hold at 0% B. Retention times for ¹⁴C-labelled compoundsare as follows; acetic acid, 5.1 min; β-hydroxybutyric acid, 7.5 min;β-hydroxyvaleric acid, 11.7 min; and β-hydroxycaproic acid, 15.3 min.Calculation of the percent turnover of 1-¹⁴C-acetyl-CoA to product wasbased on the ¹⁴C-peak integration, correcting for the double label whichoccurs in β-hydroxybutyric acid (i.e., from two molecules of1-¹⁴C-acetyl-CoA). The results are shown in Table 5.

TABLE 5 Substrate Specificity of A. eutrophus PhbA and BktBβ-Ketothiolases in the Condensation Reaction with Acetyl-CoA plusPropionyl-CoA or Butyryl-CoA¹ Enzyme Propionyl-CoA % Turnover C4 %Turnover % Turnover Sample (mM) Product C5 Product C6 Product PhbA 016.9 PhbA 0.6 13.5 0 PhbA 1.2 10.3 0 PhbA 2.4 7.0 0 BktB 0 4.2 BktB 0.61.8 13.2 BktB 1.2 1.0 15.6 BktB 2.4 0.7 17.8 Butyryl- CoA (mM) BktB 0.61.0 4.9 BktB 1.2 0.6 7.3 BktB 2.4 0.4 7.9 ¹For all reactions, theacetyl-CoA concentration was 0.30 mM (0.15 μCi ¹⁴C-acetyl-CoA per 0.50ml reaction). The concentration of propionyl-CoA or butyryl-CoA refersto the concentration in the assay. % turnover to product refers to theamount of acetyl-CoA converted to C4, C5, or C6 product. PhbAβ-ketothiolase was not tested for C6 product formation with butyryl-CoA.Blank spaces indicate products that were not detected in the experiment,probably because the #appropriate acyl-CoAs were not present in theindividual reaction mixture.

The data in Table 5 clearly demonstrate that the β-ketothiolase activityof PhbA is restricted to the catalytic formation of the C4 compoundacetoacetyl-CoA. These results can explain the metabolic block toP(3HB-co-3HV) copolymer production in recombinant E. coli containingphbA, phbB, phbC and E. coli ilvA466 (L481F) (Table 3). The data inTable 5 for A. eutrophus PhbA contrast with the suggestions of Haywoodet al. (1988b) that propose, based on thiolysis data, that the activityof PhbA β-ketothiolase alone can account for the β-ketovaleryl-CoAneeded for the C5 component in P(3HB-co-3HV) copolymer production. Thedata presented herein demonstrate that PhbA cannot efficiently catalyzethe formation of the C5 monomer. Thus, a β-ketothiolase other than PhbAappears to be involved in the production of P(3HB-co-3HV) copolymer inA. eutrophus.

As shown in Table 5, the A. eutrophus BktB enzyme catalyzes theproduction of C4, C5, and C6 β-ketoacyl-CoA compounds, with an apparentpreference for the formation of the C5 product. These results suggestthat the biochemical pathway of P(3HB-co-3HV) copolymer production in A.eutrophus involves the BktB β-ketothiolase and/or another β-ketothiolasecapable of forming the CS product. These results further suggest thatthe biosynthesis of significant amounts of P(3HB-co-3HV) copolymer inheterologous host bacteria and plants can be facilitated by the use of aβ-ketothiolase having condensation substrate specificity similar oridentical to that of BktB, i.e., being capable of condensing acetyl-CoAwith propionyl-CoA to form β-ketovaleryl-CoA.

In addition to its ability to catalyze the formation ofβ-ketovaleryl-CoA, the BktB β-ketothiolase can also catalyze theformation of β-keto-caproyl-CoA (Table 5). Based on this observation,the BktB β-ketothiolase can also be utilized for the production ofcopolymers containing monomers ranging in size from C4 to C6. Althoughthe condensing activity of the BktB β-ketothiolase beyond C6 has notbeen tested, it is possible that this enzyme can catalyze the formationof β-ketoacyl-CoA compounds greater than C6. It is likely that plant andbacterial cells containing BktB and also expressing appropriateβ-ketoacyl-CoA reductases and PHA synthases capable of utilizing therange of β-ketoacyl-CoA substrates produced by BktB (for example,Nocardia corallina PHA synthase; Dennis, 1994) can produce copolymerscontaining monomer units ranging from C4 to C6, and possibly beyond. Oneexample is a copolymer comprising β-hydroxy-butyrate (3HB) andβ-hydroxycaproate (3HC), designated P(3HB-co-3HC) copolymer.

Thiolysis Activity of A. eutrophus BktB

To enhance further our understanding of the kinetic characteristics ofBktB β-ketothiolase, a thorough kinetic analysis of the enzyme'sthiolysis activity was conducted using purified protein. The assay todetermine the kinetic parameters K_(m) and V_(max) for β-ketobutyryl-CoA(C4), β-ketovaleryl-CoA (C5), and β-ketocaproyl-CoA (C6), was performedat 25° C. by the in situ generation of the β-keto compounds from theircorresponding trans-2,3-enoyl-CoAs essentially as described by Haywoodet al. (1988). Utilization of variable substrate concentrationspermitted the determination of the descriptive kinetic parameters of theβ-ketothiolase. Kinetic rates were determined only at fixed substrateconcentrations in the Haywood et al. communication.

Purified BktB protein was prepared by growing E. coli EE36 transformedwith pMON25754 in LB broth containing ampicillin (100 μg/ml) overnightat 37° C. Cells were then harvested by centrifugation, and the pelletwas resuspended in 50 mM KPi and 5% glycerol, sonicated, and thesonicate centrifuged to remove cellular debris. The cell extract wasdesalted using a PD-10 column (Pharmacia), and proteins resolved using aPharmacia FPLC system and MonoQ anion-exchange column (Pharmacia).Buffer A was 10 mM Tris buffer, pH 7.8, plus In M DTT; buffer B wasbuffer A plus 1 M KCl. The gradient was 0 to 10% B in 10 min, then to35% B in 40 min. The flow rate was 1 ml/min, and the resolved proteinswere collected at the rate of 1 ml/fraction. The BktB protein was foundin fractions 20 to 27, with the maximum activity peak at fraction 23.Fraction 23 used for N-terminal sequencing was desalted, washed, andstored in 10 mM sodium bicarbonate using a Centricon-10 concentrator(Amicon, Inc.). Protein was found to be pure according to SDS-PAGEanalysis, with an apparent molecular weight of 39.5 kD (data not shown).The enzyme was stored in 10 mM Tris-HCl, pH 7.8, 1 mM DTT, 50% glycerolat −20 ° C. Activity remained constant for a minimum of two months underthese conditions.

The purified BktB β-ketothiolase was diluted to approximately 1 mg/ml in10 mM Tris-HCl, pH 7.8, 50% glycerol. Two rabbits were boosted initiallywith 200 jig of purified protein in a total volume of 1 ml by ScientificAssociates Inc. (St. Louis, Mo.). Bleeds after the first boost yieldedpolyclonal antibodies which produced a single immunoreactive band atapproximately 45 kD on Western blots containing a range of dilutions ofpurified BktB. The detection limit of the antibodies was determined tobe 1 ng. These antibodies can be used to detect proteins having similarimmunoreactive properties to BktB β-ketothiolase, thus making itpossible to detect ketothiolases having similar enzymatic activity toBktB in crude extracts.

The C5 and C6 enoyl-CoAs were synthesized according to Schulz (1974) andpurified using semi-prep C8-reverse-phase HPLC as described above forβ-ketovaleryl-CoA. β-ketobutyryl-CoA (acetoacetyl-CoA) was purchasedfrom Sigma (St. Louis, Mo.). The assay mixture (1 ml final volume)contained 150 mM EPPS, pH 8.0, 50 mM MgCl₂, 1.5 mM pyruvate, 0.4 mMNAD⁺, and a coupling enzyme cocktail of three units crotonase, fiveunits β-hydroxyacyl-CoA dehydrogenase, and 10 units lacticdehydrogenase. The enoyl-CoA was added to this mixture and theabsorbance et 304 nm was monitored until it reached a plateau (typicallyabout 5 min). CoA was added to this mixture, followed by BktBβ-ketothiolase to initiate the reaction. The decrease in absorbance at304 nm due to the thiolysis of the β-ketoacyl-CoA was utilized as adirect measure of activity. Extinction coefficients to convertabsorbance units to μmol of product were 19.5, 12.2, and 14.0 cm⁻¹mM⁻¹for β-ketobutyryl-CoA, β-ketovaleryl-CoA, and β-ketocaproyl-CoA,respectively. β-ketoacyl-CoA concentration was varied from 5 to 80 μM(CoA was fixed at 200 μM), and CoA concentration was varied from 20 to400 μM (β-ketocaproyl-CoA was fixed at 60 μM) for determining kineticparameters for β-ketoacyl-CoA and CoA, respectively. Reaction rates werecalculated in the steady-state within the first minute followinginitiation. All data were fitted to the normal Michaelis-Menten equationusing a non-linear regression analysis. The results are shown in Table6.

TABLE 6 Kinetic Parameters for BktB β-Ketothiolase in the ThiolysisReaction Substrate K_(m) (μM) V_(max) (u/mg) V_(max)/K_(m)B-ketobutyryl-CoA² 37 ± 3 122 ± 5 3.0 B-ketovaleryl-CoA² 15 ± 1 236 ± 816 B-ketocaproyl-CoA²  6.0 ± 0.7 103 ± 3 17 CoA³ 53 ± 6  98 ± 3 1.8 ¹Aunit, u, refers to μmol product formed per minute. ²Kinetic parameterswere determined using a fixed concentration of CoA of 200 μM. ³Kineticparameters were determined using a fixed concentraiton ofβ-keto-caproyl-CoA of 60 μM.

The results in Table 6 show that β-ketovaleryl-CoA can be turned over byBktB by about a factor of two faster than the other two substratesaccording to the V_(max) values. This faster turnover is somewhatmodulated by a higher K_(m) compared to β-ketocaproyl-CoA. Overall,based on V_(max)/K_(m), the enzyme utilizes C5 and C6 substrates aboutequivalently, whereas β-keto-butyryl-CoA is approximately five-fold lessefficient in catalytic turnover. These results are consistent with thecondensation data for BktB shown in Table 5. From this, and incombination with the condensation and thiolysis results on PhbAβ-ketothiolase, one might hypothesize that gathering thiolysis dataalone for a particular β-keto-thiolase would be sufficient to suggestthe activity of the enzyme in the condensation direction. However, asshown below, this is not necessarily true.

Thiolysis and Condensation Activity of Other β-Ketothiolases

Two additional β-ketothiolases from A. eutrophus (cosmid clones AE65 andAE902; Slater et. al., 1988), plus two β-ketothiolases from Zoogloearamigera, were analyzed for thiolysis and condensation activity usingthe assays described above. Zoogloea ramigera (ATCC 19623) was grown at30° C. for 48 hours in medium containing; 9.4 g/l K₂HPO₄; 2.2 g/l KH₂PO₄; 2.5 g/l MgSO₄•7H₂O; 3 g/l N-Z Amine; and 10 g/l Na-gluconate. Ecoli containing cosmid clones AE65 and AE902 were grown for 16 hours at37° C. in LB supplemented with 50 μg/ml kanamycin. For the thiolysisassay, activity was monitored at a fixed concentration of β-ketoacyl-CoAat 40 μM and CoA at 100 μM. All of the protein extracts were partiallypurified using the Pharmacia FPLC MonoQ separation described above.Table 7 shows the thiolysis results.

TABLE 7 Substrate Specificity of Various β-Ketothiolases in theThiolysis Reaction¹ β-ketobutyryl-CoA β-ketovaleryl-CoAβ-ketocaproyl-CoA Enzyme Sample (u/mg) (u/mg) (u/mg) Z ramigera (A)²0.41 0.15 0.02 Z ramigera (B)² 0.21 0.19 0.11 pAE65 0.156 0.502 3.16pAE902 0.585 1.25 0.561 ¹Activity was determined using partiallypurified enzymes. The concentration of substrate was 40 μM. pAEdesignations are A. eutrophus cosmid clones. ²The Z ramigera proteinsample yielded two active ketothiolase peaks upon FPLC MonoQ resolution,designated A and B.

MonoQ separation of the Z. ramigera protein extract resulted in tworesolved active β-ketothiolase peaks. The first peak, designated A,shows thiolysis activity with C4 and C5 substrate, but little if anywith C6. Based on this activity, this β-ketothiolase in all likelihoodis the enzyme purified and described by Davis et al., (1987), and is theprotein expressed specifically for PHB production in this organism. Zramigera peak B, which has not been previously described, appears tohave broadened specificity, at least to C6.

A. eutrophus cosmid clones pAE65 and pAE902, when similarly analyzed forthiolysis activity, both showed specificity at least to C6. Clone pAE65,in fact, likely has significant activity with β-ketoacyl-CoAs of higherchain-length then C6 based on the significant increase in activity whengoing from C4 to C6. Additional results (not shown) for pAE65demonstrate dehydratase and β-keto-acyl-CoA dehydrogenase activity inthe same resolved MonoQ fraction. This suggests that the pAE65 clonecontains the genes for the fatty acid β-oxidative complex proteins of A.eutrophus. Interestingly, clone pAE902 encodes an enzyme havingthiolysis activity with β-ketoacyl-CoA substrates ranging from C4 to C6similar to that of BktB. As such, considering only the tholysis resultsin Table 7, pAE902 might be considered a good candidate forβ-ketovaleryl-CoA condensation production from acetyl-CoA andpropionyl-CoA. However, it is important to consider condensationdirection activity as well. Condensation results for this clone, alongwith that of the other β-ketothiolases, are shown in Table 8.

TABLE 8 Substrate Specificity of Various β-Ketothiolases in theCondensation Reaction with Acetyl-CoA Plus Propionyl-CoA or Butyryl-CoA% Turnover % Turnover % Turnover Enzyme Sample Substrates¹ C4 Product C5Product C6 Product Z. ramigera (A)² C2 23.3 Z. ramigera (A)² C2 + C312.4 7.1 Z. ramigera (A)² C2 + C4 18.9 0 Z. ramigera (B)² C2 3.4 Z.ramigera (B)² C2 + C3 1.1 2.7 Z. ramigera (B)² C2 + C4 1.2 2.6 pAB65 C25.7 pAE65 C2 + C3 2.2 58.6 pAE65 C2 + C4 0.5 84.3 pAE902 C2 0 pAE902C2 + C3 0 0 pAE902 C2 + C4 0 0 ¹For all reactions, the acetyl-CoA (C2)concentration was 0.40 mM (0.15 μCi¹⁴C-acetyl-CoA per 0.50 ml reaction).The concentration of propionyl-CoA (C3) or butyryl-CoA (C4) was 2.0 mMwhen present. % turnover to product refers to the amount of acetyl-CoAconverted to C4, C5, or C6 product. pAE designations are A. eutrophuscosmid clones. ²The Z. ramigera protein sample yielded two activeketothiolase peaks upon FPLC Mono-Q resolution, designated A and B.

The data presented in Table 8 demonstrate condensation activity for allsamples tested, excluding pAE902. Consistent with the pattern shown inthe thiolysis results, the Z. ramigera enzymes are capable of producingC5, and C5 and C6 condensation products, for peaks A and B,respectively. These then represent two possible alternative candidatesfor use in the biosynthesis of P(3HB-co-3HV) copolymer in recombinantsystems. Similarly, cosmid clone pAE65 shows activity that parallelsthat seen in the thiolysis assay.

The pAE902 cosmid clone showed no condensation products, and in additionexhibited greater than 50% hydrolysis of the starting ¹⁴C-acetyl-CoA inthe time-course of the reaction (hydrolysis data not shown). This resultshows that it is important to demonstrate condensation substratespecificity activity for a particular β-ketothiolase to evaluate whetheror not it is useful in producing P(3HB-Co-3HV) copolymer. That is, fromthis result with pAE902, it is not obvious that the thiolysis activityof a particular β-ketothiolase will necessarily translate to usefulinformation regarding its condensation activity. This also demonstratesthe utility of a quantitative assay such as the one described herein formeasuring condensation activity.

Cloning of the A. eutrophus bktb Gene

As noted above, Slater et. al. (1988) identified several putative A.eutrophus β-ketothiolase clones, including pBK6, by screening E. coliharboring an A. eutrophus cosmid library for β-ketothiolase activity.

The β-ketothiolase gene encoded on pBK6 was mapped by serial subcloningand deletion analysis, and sequenced (Sambrook et al., 1989). Thesequence is shown in SEQ ID NO:9. For the purpose of the presentinvention, this gene has been designated as the bktB gene of A.eutrophus. This gene was deposited under the terms of the BudapestTreaty in the American Type Culture Collection, 12301 Parklawn Drive,Rockville, Md. 20852, U.S.A., as an insert in plasmid pMON25728contained in E. coli DH5α on Mar. 6, 1996, designated ATCC 98007.

pMON25728 contains a fragment of approximately 3.9 Kb extending from aBgl II site approximately 550 bp upstream of the bktB open reading frameto a Nco I site downstream of bktB. There is a single additional Nco Isite in this fragment, and it is within bktB. The Bgl II site upstreamof bktB is the single Bgl II site shown in plasmid pBK6 (Slater et al,1988). pMON25728 contains the 3.9 Kb Bgl II-NcoI fragment in which bothsites have been end-filled and ligated to DNA of pBluescipt KS+(Stratagene). pBluescript KS+ polylinker sites were cut with Sac I andEco RV, and the Sac I site was treated with exonuclease to create ablunt end. The Bgl II site of the A. eutrophus DNA has been fused to theEcoRV site of pBluescript KS+.

Automated Edman degradation chemistry was used to determine theNH₂-terminal protein sequence (Hunkapiller et al., 1983) of purifiedBktB protein. A Perkin Elmer Applied Biosystems Division sequencer(Foster City, Calif.) was employed for the analysis. The respectivePTH-aa derivatives were identified by RP-HPLC analysis in an on-linefashion employing a Brownlee 2.1 mm I.D. PTH-C 18 column.

Amino acid sequencing of the protein purified (0.9 mg/ml) as describedabove revealed that the N-terminal sequence minus the initiatingmethionine (TREVVVVSGVRTAIG, SEQ ID NO:10) corresponds to that predictedby the DNA sequence shown in SEQ ID NO:9, as well as the deduced aminoacid sequence shown in SEQ ID NO: 11.

P(3HB-co-3HV) Copolymer Production and Active Expression of bktB in E.coli

The data reported in Table 3 (Example 2) suggested the presence of ametabolic block to P(3HB-co-3HV) copolymer production in E. colitransformed with the A. eutrophus phbA, phbB, phbC, and E. coli ilvA466(L481F) genes. To determine whether the expression of the A. eutrophusbktB gene in E. coli is catalytically effective in P(3HB-co-3HV)copolymer biosynthesis in vivo, the following experiment was performed.

Two sets of E. coli DH5α cells were transformed with the plasmidcombinations shown in Table 9 as described in Example 2 in the sectionentitled “In vivo Analysis of Cloned E. coli ilvA genes”:

TABLE 9 Plasmid Combinations Used for the Production of P(3HB-co-3HV)Copolymer in Transformed E. coli Antibiotic Gene resistance PromoterOrigin of replication Set 1 pMON25628 phbB Spectinomycin recA ori327pMON25629 phbC Kanamycin tac ori pACYC pMON25636 phbA Ampicillin tac oriCoLEl Set 2 pMON25628 phbB Spectinomycin recA ori327 pMON25629 phbCKanamycin tac ori pACYC pMON25728 bktB Ampicillin native ori CoLEl

Maps of pMON25628, pMON25629, and pMON25636 are shown in FIGS. 20-22,respectively. pMON25728, a derivative of pBK6 (Slater et al., 1988), wascreated by first subcloning a 5.5 Kb NotI/EcoRI fragment of pBK6 intopBluescript KS+(Stratagene), creating pMON25722. pMON25722 wassubsequently digested with BglII and EcoRI, releasing a 1.1 Kb fragment.The remaining vector was blunt ended with Kienow and religated, creatingpMON25724. Finally, pMON25724 was partially digested with NcoI, anddigested to completion with SacI, releasing a 0.9 Kb fragment Theremaining vector was blunt ended with KIenow and religated, creatingpMON25728.

Each set of transformed cells containing all three plasmids was selectedby the addition of all three antibiotics (ampicillin, 100 μg/ml;kanamycin, 50 i g/ml; spectinomycin, 75 μg/ml) to the growth medium (LBagar). Single isolated colonies were grown on LB medium containing allthree antibiotics to early log phase and induced with IPTG and nalidixicacid. Cells were pelleted by centrifugation, resuspended in 500 μl of 50mM KPi/5% glycerol, and sonicated on ice. Enzyme assays as describedabove were performed on the selected cells to confirm expression andactivity of the reductase (PhbB), and, in the thiolysis direction, theβ-ketothiolases (PhbA and BktB). The results are shown in Table 10.

TABLE 10 Activity of PhbA, BktB, and PhbB in Transformed E. coli DH5αThiolase Rate Thiolase Rate Reductase Rate (AU/min³) (AU/min³) (AU/min³)Gene C4 substrate¹ C5 substrate² C4 substrate¹ Set 1 pMON25628 phbB0.035 pMON25629 phbC pMON25636 phbA 0.330 0.013 Set 2 pMON25628 phbB0.051 pMON25629 phbC pMON25728 bktB 0.132 0.135 Control E. coli DH5α0.008 0.004 ¹C4 substrate: acetoacetyl-CoA ²C5 substrate:β-ketovaleryl-CoA ³AU refers to absorbance units

The activities of the PhbA and BktB enzymes were as expected based uponthe kinetic data reported in Table 5, i.e., the PhbA β-ketothiolase wasnot avtive in thiolysis of the C5 substrate, while the BktBβ-ketothiolase exhibited significant activity with CS substrate. Thelevels of reductase (PhbB) activity were low due the nature of the E.coli DH5α host cells. Nalidixic acid is commonly used as a chemicalinducer of the SOS response of E. coli (Walker, 1987). Since the phbBgene is transcribed from the SOS responsive recA promoter, and since E.coli DH5α are recA, there was no induction from the RecA promoter uponthe addition of nalidixic acid. The low levels of reductase activityobserved are due to leaky expression from the recA promoter. PhbCactivity was not measured enzymatically; instead, the production of PHAwithin the E coli cells was taken as proof of active PhbC enzyme.

E. coli DH5α cells contining both sets of plasmids expressing all of therequired PHA biosynthesis enzymes were grown on 50 ml of LB brothcontaining all three antibiotics supplemented with 0.75% glucose and0.15% propionate. The tac promoters were induced with IPTG at a finalconcentration of 0.5 mM. Nalidixic acid was added to a finalconcentration of 60 μg/ml. The cells were grown under antibioticselection (see above) for 24 hours and assayed for the presence of PHApolymer. Cells were centrifuged at 7,000 rpm for 20 min., washed with 25ml methanol, recentrifuged, washed with 15 ml hexane, and recentrifuged.Cell pellets were dried under N₂ for two hours, and the dry cell weightsdetermined. 6.5 ml of chloroform were added to extract the PHA at 100°C. for one hour. The solution was cooled and filtered through a PTFEsyringe filter (13 mm diameter, 0.45 μm pore). The PHA was precipitatedby the addition of 50 ml of methanol, centrifuged, and washed withhexane. The polymer was dried at 70° C. for two hours, and polymerweight was determined. Methanolysis was performed by dissolving 3-5 mgof PHA sample by the addition of 1 ml of CHCl₃, with methyl benzoate asan internal standard for normalization of the data. One ml of 15%H₂SO₄/MeOH was added, and the mixture was heated at 100° C. for twohours. The samples were cooled, and 0.5 ml of H₂O were added. The lowerCHCl₃ layer was removed, and excess water was removed using Na₂SO₄. Thesolution was then analyzed by gas chromatography as described in thesection entitled “in vivo Analysis of Cloned E. coli ilvA Genes”. Theresults are shown in Table 11.

TABLE 11 Gas Chromatographic Analysis of PHA Produced by Transformed E.coli DH5α Cell weight PHA weight Set (mg) (mg) % PHA % C4 % C5 Set 1(phbA + phbB 146.5 38 26 99.1 0.9 + phbC) Set 2 (BktB + phbB 132.4 32 2495 5 + phbC)

The results in Table 11 demonstrate that the total amount ofP(3HB-co-3HV) copolymer produced in cells transformed with both sets ofplasmid vector combinations was comparable. Furthermore, cellsexpressing the BktB β-keto-thiolase produced a greater percentage(five-to-six-fold) of the C5 monomer than cells expressing the PhbAβ-ketothiolase. In addition, the formation of P(3HB-co-3HV) in theexperiments reported in Table 1 1 was confirmed by ¹H-NMR (data notshown). These results support the hypothesis that the A. eutrophus PhbAβ-ketothiolase was responsible for the metabolic block preventing theproduction of significant C5 constituent of the copolymer reported inTable 3.

Other β-Ketothiolases, β-Ketoacyl-CoA Reductases, and PHA Synthases forthe Production of P(3HB-co-3HV) Copolymer

Examples of enzymes useful in the production of P(3HB-co-3HV) copolymerhave been described above. However, the present invention is not limitedthereto, and other β-ketothiolases, β-ketoacyl-CoA reductases, and PHAsynthases that can be used in the present invention exist, or can beobtained from other sources.

One source includes organisms possessing useful PHA biosyntheticenzymes. These include, for example, Alcaligenes eutrophus, Alcaligenesfaecalis, Aphanothece sp., Azotobacter vinelandii, Bacillus cereus,Bacillus megaterium, Beyierinkia indica, Derxia gummosa,Methylobacterium sp., Microcoleus sp., Nocardia corallina, Pseudomonascepacia, Pseudomonas extorquens, Pseudomonas oleovorans, Rhodobactersphaeroides, Rhodobacter capsulatus, Rhodospirillum rubrum (Brandl etal., 1990; Doi, 1990), and Thiocapsa pfennigii. Using the methodsdescribed herein, one can identify and isolate DNAs encoding otherβ-ketothiolases, β-ketoacyl-CoA reductases, and PHA synthaeses useful inthe present invention from these and other organisms capable ofproducing PHAs.

The present invention encompasses not only the A. eutrophus DNA sequenceshown in SEQ ID NO:9, but also biologically functional equivalentnucleotide sequences. The phrase “biologically functional equivalentnucleotide sequences” denotes DNAs and RNAs, including chromosomal DNA,plasmid DNA, cDNA, synthetic DNA, and mRNA nucleotide sequences, thatencode peptides, polypeptides, and proteins exhibiting the same orsimilar β-keto-thiolase enzymatic activity as that of A. eutrophus BktBwhen assayed enzymatically or by complementation. Such biologicallyfunctional equivalent nucleotide sequences can encode peptides,polypeptides, and proteins that contain a region or moiety exhibitingsequence similarity to the corresponding region or moiety of the A.eutrophus BktB β-ketothiolase.

Thus, one can isolate enzymes useful in the present invention fromvarious organisms based on homology or sequence identity. Tombolini etal. (1995) conducted a comparative study of the homologies of knownβ-keto-thiolases and reductases. Although one embodiment of a nucleotidesequence encoding A. eutrophus bktB is shown in SEQ ID NO:9, it shouldbe understood that other biologically functional equivalent forms of A.eutrophus BktB-encoding nucleic acids can be readily isolated usingconventional DNA-DNA or DNA-RNA hybridization techniques. Thus, thepresent invention also includes nucleotide sequences that hybridize toSEQ ID NO:9 and its complementary sequence, and that code on expressionfor peptides, polypeptides, and proteins exhibiting the same or similarenzymatic activity as that of A. eutrophus BktB β-ketothiolase. Suchnucleotide sequences preferably hybridize to SEQ ID NO:9 or itscomplementary sequence under moderate to high stringency (see Sambrooket al., 1989). Exemplary conditions include initial hybridization in 6×SSC, 5× Denhardt's solution, 100 mg/ml fish sperm DNA, 0.1% SDS, at 55°C. for sufficient time to permit hybridization (e.g., several hours toovernight), followed by washing two times for 15 min each in 2× SSC,0.1% SDS, at room temperature, and two times for 15 min each in 0.5-1 ×SSC, 0.1% SDS, at 55° C., followed by autoradiography. Typically, thenucleic acid molecule is capable of hybridizing when the hybridizationmixture is washed at least one time in 0.1× SSC at 55° C., preferably at60° C., and more preferably at 65° C.

The present invention also encompasses nucleotide sequences thathybridize under salt and temperature conditions equivalent to thosedescribed above to genomic DNA, plasmid DNA, cDNA, or synthetic DNAmolecules that encode the amino acid sequence of A. eutrophus BktBβ-ketothiolase, and genetically degenerate forms thereof due to thedegenerancy of the genetic code, and that code on expression for apeptide, polypeptide, or protein that has the same or similarketothiolase enzymatic activity as that of A. eutrophus BktBβ-ketothiolase.

Biologically functional equivalent nucleotide sequences of the presentinvention also include nucleotide sequences that encode conservativeamino acid changes within the A. eutrophus BktB amino acid sequence,producing silent changes therein. Such nucleotide sequences thus containcorresponding base substitutions based upon the genetic code compared towild-type nucleotide sequences encoding A. eutrophus BktB.

In addition to nucleotide sequences encoding conservative amino acidchanges within the naturally occurring A. eutrophus BktB amino acidsequence, biologically functional equivalent nucleotide sequences of thepresent invention also include genomic DNA, plasmid DNA, cDNA, syntheticDNA, and mRNA nucleotide sequences encoding non-conservative amino acidsubstitutions, additions, or deletions. These include nucleic acids thatcontain the same inherent genetic information as that contained in theDNA of SEQ ID NO:9, and which encode peptides, polypeptides, or proteinsexhibiting the same or similar β-ketothiolase enzymatic activity as thatof A. eutrophus BktB. Such nucleotide sequences can encode fragments orvariants of A. eutrophus BktB. The A. eutrophus BktB β-ketothiolase-likeenzymatic activity of such fragments and variants can be identified bycomplementation or enzymatic assays as described above. Thesebiologically functional equivalent nucleotide sequences can possess from40% sequence identity, or from 60% sequence identity, or from 80%sequence identity, to 100% sequence identity to naturally occurring DNAor cDNA encoding A. eutrophus BktB -ketothiolase, or correspondingregions or moieties thereof. However, regardless of the percent sequenceidentity of these biologically functional equivalent nucleotidesequences, the encoded proteins would possess the same or similarenzymatic activity as that of BktB β-ketothiolase. Thus, thebiologically functional equivalent nucleotide sequences encompassed bythe present invention include sequences having less than 40% sequenceidentity to SEQ ID NO:9, so long as they encode peptides, polypeptides,or proteins having the same or similar enzymatic activity as that ofBktB β-ketothiolase.

Mutations made in A. eutrophus BktB β-ketothiolase cDNA, chromosomalDNA, plasmid DNA, synthetic DNA, mRNA, or other nucleic acid preferablypreserve the reading frame of the coding sequence. Furthermore, thesemutations preferably do not create complementary regions that couldhybridize to produce secondary mRNA structures, such as loops orhairpins, that would adversely affect mRNA translation.

Useful biologically functional equivalent forms of the DNA of SEQ IDNO:9 include DNAs comprising nucleotide sequences that exhibit a levelof sequence identity to corresponding regions or moieties of the genomicDNA of SEQ ID NO:9 from 40% sequence identity, or from 60% sequenceidentity, or from 80% sequence identity, to 100% sequence identity.However, regardless of the percent sequence identity of these nucleotidesequences, the encoded peptides, polypeptides, or proteins would possessthe same or similar enzymatic activity BktB β-ketothiolase. Thus,biologically functional equivalent nucleotide sequences encompassed bythe present invention include sequences having less than 40% sequenceidentity to SEQ ID NO:9, so long as they encode peptides, polypeptides,or proteins having the same or similar enzymatic activity as BktBβ-ketothiolase. Sequence identity can be determined, for example, usingthe “BestFit,” “Gap,” or “FASTA” programs of the Sequence AnalysisSoftware Package, Genetics Computer Group, Inc., University of WisconsinBiotechnology Center, Madison, Wis. 53711, or using the “BLAST” program(Altschul et al., 1990).

Useful biologically functional equivalent forms of the BktB proteinsequence shown in SEQ ID NO:11 include polypeptides and proteinscomprising amino acid sequences that exhibit a level of sequenceidentitiy to the BktB protein or regions thereof of at least about 25%,preferably at least about 30%, and more preferably at least about 35%.For this purpose, sequence identity can be determined using the “TFASTA”program of the Sequence Analysis Software Packgage referred to above, orby the “BLASTP” program of Altschul et al., 1990, for example. Usefulbiologically functional equivalent forms of the BktB protein of thepresent invention also include polypeptides and proteins wherein regionsrequired for BktB-like catalytic activity are essentially conserved orretained, but wherein non-critical regions may be modified by amino acidsubstitutions, deletions, or additions.

Due to the degeneracy of the genetic code, i.e., the existence of morethan one codon for most of the amino acids naturally occuring inproteins, genetically degenerate DNA (and RNA) sequences that containthe same essential genetic information as the DNA of SEQ ID NO:9 of thepresent invention, and which encode the same amino acid sequence as thatof A. eutrophus BktB, are encompassed by the present invention.Genetically degenerate forms of any of the other nucleic acid sequencesdiscussed herein are encompassed by the present invention as well.

The nucleotide sequences described above are considered to possess abiological function substantially equivalent to that of the A. eutrophusBktB gene of the present invention if they encode peptides,polypeptides, or proteins having β-ketothiolase enzymatic activitydiffering from that of A. eutrophus BktB by about +30% or less,preferably by about ±20% or less, and more preferably by about ±10% orless when assayed in vivo by complementation or by the enzymatic assaysdiscussed above.

Based on amino acid sequence homology or sequence identity, one mightattempt to predict the function of a protein of interest; however, thismay not accurately reflect the activity of the protein, and it wouldtherefore be necessary to assay the β-ketothiolase enzymatic activitythereof.

Other β-ketothiolases useful in the present invention may be derivedfrom enzymes involved in lipid biosynthesis and β-oxidation which arenot directly involved in PHA production.

EXAMPLE 9 Production of PHBV in E. coli Expressing ilvA and bktB

Genes required for the pathway diagrammed in FIG. 3 were assembled inrecombinant E. coli in order to test the feasibility of producing PHBVfrom threonine in a pathway utilizing ilvA and bktB (both discussedabove). E. coli DH12s (obtained from Gibco/BRL) was transformed withpMON25779 (harboring A. eutrophus phbB and phbC under LacI control) andpMON25822, which expresses bktB from the A. eutrophus bktB promoter. Theresulting recombinant E. coli strain expresses BktB β-ketothiolase, PhbBβ-ketoacyl-CoA reductase, and PhbC PHA synthase.

pMON25779 was constructed as follows. pJM9131 (Kidwell et al., 1995) wasdigested with Stu I and the vector fragment was re-closed, therebydeleting a fragment of appoximately 1 Kb that is required for productionof the PhbA β-ketothiolase. The resulting plasmid, designated pMON25748,was digested with Bsa AI and Eco RI, and the fragment encoding a portionof phbC and the entire phbB gene was used to replace the Bsa AI-Eco RIfragment encoding a portion of phbC in pMON25629 (FIG. 21), producingpMON25779. pMON25779 encodes phbCB under Ptac promoter control.

pMON25822 (FIG. 25) contains a fragment of apporoximately 1.8 Kbencoding A. eutrophus bktB cloned into in the vector pBBRIMCS-3 (Kovachet al., 1994). pMON25822 was constructed as follows. pMON25765 is apBluescript KS+ derivative that contains A 1.8 Kb Fragment encodingbktB. This plasmid is constructed by digesting pMON25728 (discussedabove) partially with Sal I, then completely with Xho I, and cloning theresulting 1.8 Kb fragment into pBluescript KS+ that has been digestedwith Sal I and Xho I. The 1.8 Kb bktB fragment contains a total of 3 SalI sites. pMON25765 was digested with Xho I and Xbal, and the 1.8 Kbfragment encoding bktB was ligated to pBBRI MCS-3 digested with Xho Iand Xbal.

To the strain harboring pMON25779 and pMON25822 was added either pSE280(an expression vector lacking an ilvA gene: Brosius, 1989) or pMON25683(expressing ilvA466 from the LacI-regulated promoter of pSE280: FIG.13). Each strain was grown to late log phase in 50 ml LB containingappropriate antibiotics, then the cells were pelleted by centrifugation,washed 2 times with M9 minimal salts, and resuspended in 100 ml M9minimal media containing 0.5% glucose as the carbon source. Theresuspended cells were split into two, 50 ml cultures, and threonine wasadded to one flask from each pair to a final concentration of 25 mM. Theflasks were incubated overnight at 37° C. with vigorous shaking. Afterabout 18 hours, the cells were concentrated by centrifugation, andpolymer composition was analyzed as described above. The results areshown in Table 12.

TABLE 12 Production of PHBV from glucose in E. coli expressing ilvA andbktB plasmid ilvA allele¹ Threonine added? mol% hydroxyvalerate none Y3.5 none N N.D.² ilvA466³ Y 31.4  ilvA466³ N 7.9 ¹All strains arechromosomally ilvA⁺. ²N.D. = none detected. ³ilvA466 contains the L481Fmutation.

As demonstrated in Table 12, E. coli harboring ilvA466 and bktB can makePHBV, even in the absence of added threonine. E. coli lacking a plasmidexpressing ilvA466 also produced some PHBV if threonine was added to themedia. In this case, conversion of threonine to α-ketobutyratepresumably proceeds via the chromosomally-encoded IlvA protein.

These data demonstrate that substitution of bktB for phbA overcomes themetabolic block to PHBV formation in E. coli expressing only the phboperon (Table 3). These data also demonstrate that the PHBV copolymercan be produced in vivo from endogenous substrate pools if the cellexpresses a partially-deregulated threonine deaminase and PHAbiosynthesis enzymes having appropriate substrate flexibility.

EXAMPLE 10 Production of P(3HB) in Seeds of Canola and Soybean

Poirier et al. (1992) and Nawrath et al. (1994) demonstrated theproduction of poly(3-hydroxybutyrate) (P(3HB)) homopolymer in leaves oftransgenic Arabidopsis plants. Although this was the first demonstrationof a plant-produced PHA, Arabidopsis is not an attractive commercialcandidate for the large scale production of PHAs as this plant isprimarily used as a research tool, and has no agronomic value. However,crops such as canola and soybean, for example, are excellent candidatesfor commercial PHA production in view of the vast acreage upon whichthey are grown. In addition, these crops are used for vegetable oilproduction, and significant pools of acetyl-CoA are present to supportthe oil biosynthesis that occurs in the seeds. As noted earlier,plant-produced PHAs have the potential to lower the current high cost ofthe microbially-produced PHAs by eliminating the fermentation costs andthe expensive feedstocks required by microorganisms. For example, withproperly timed and targeted expression of the PHB biosynthetic enzymesin seed plastids, one would expect to produce high levels (100/M20%fresh seed weight) of PHB polymer. Described below are the firstdefinitive experimental results demonstrating PHA production incommercially useful plants, such as canola and soybean.

Transformation Vectors

Two Agrobacterium plant transformation vectors were constructed tointroduce the entire PHB biosynthetic pathway from A. eutrophus intocanola and soybean. The first plasmid, pMON25626 (FIG. 23), comprisesthe genetic information for the expression of three genes in thetransformed plant: phbC,phbB and CP4 EPSPS. Specifically, pMON25626contains, begining at the right border (RB) and continuing clockwise:the 7S β-conglycinin seed-specific promoter (Doyle et al., 1986;Slighton and Beachy, 1987) followed by the Arabidopsis small subunit ofRUBP carboxylase chloroplast transit peptide (Arab-SSU1A; Stark et al.,1992) translationally fused via an Nco I site to the phbC gene, followedby the polyadenylation and transcription termination signal contained inE 9 3′ (Coruzzi, et al., 1984; Morelli et al., 1985). The vectorcontinues with the 7S promoter, Arab-SSI1A, phbB, and E9 3′ terminationsignal, constructed as above. Additionally, pMON25626 contains the FMVpromoter (Richine et al., 1987), followed by the petunia EPSPS(5-enol-pyruvylshikimate-3-phosphate synthase) chloroplast transitpeptide (PEPSP; Shah et al., 1986) translationally fused to the CP4EPSPS gene (PCT International Publication WO 92/04449; Padgette et al.,1996), and the nos 3′ polyadenylation and transcription terminationsignal (Fraley et al., 1983). The CP4 EPSPS gene was used as theselectable herbicide resistance marker (“glyphosate selection”) in thetransformation procedure (Shah et al., 1986). The CP4 EPSPS enzyme is aglyphosate-resistant form of the EPSPS enzyme involved in aromatic aminobiosynthesis in plants and bacteria which catalyzes the reversiblereaction of shikimate-3-phosphate and phosphoenolpyruvate to produce5-enolpyruvylshikimate-3-phosphate (Padgette et al., 1996).

The second plant transformation vector, pMON25638 (FIG. 24), containingthe phbA and the CP4 EPSPS genes, was constructed in a mannersimilar tothat of pMON25626. The promoters, chloroplast transit peptides, andpolyadenylation and transcription termination signals were identical tothose described above for pMON25626. Both pMON25626 and pMON25638contain the identical border sequence and replication origins andfunctions as described for pMON25668 of Example 5 in the sectionentitled “Expression and Activity in Transformed Soybean Callus”.

Transformation Protocols

pMON25626 and pMON25638 were introduced into Agrobacterium tumefaciensABI (Koncz and Schell, 1986) by triparental mating (Ditta et al., 1980),which was then used for the transformation of canola (Fry et al., 1987;Radke et al., 1988) and soybean (Hinchee et al., 1988), with themodifications indicated below.

Canola Transformation

Plant Material

Stock plants were produced from seeds of the Westar variety planted inMetro Mix 350 and germinated in a growth chamber under a day temperatureof 15° C., a night temperature of 10° C., a 16 hour day/8 hour nightillumination period, a light intensity of 600 μEn m-²s⁻¹, and 50%relative humidity. Seedlings were subirrigated with water daily, andsoaked with a 15-30-15 nutrient solution every other day for one hour.At three weeks, seedlings were transferred into 6″ pots. Five week oldplants were harvested once the plants bolted, but prior to flowering(plants with up to three flowers can be employed, however). The leavesand buds were removed from the stem, and the 4-5 inches of stem justbelow the flower buds were used as the explant tissue source. Just priorto inoculation, the stems were sterilized by soaking in 70% ethanol for1 min, 38% Chlorox (4% sodium hypochlorite) for 20 min, rinsing twotimes in sterile deionized water, and soaking in two tablespoons ofCaptan (Captan 50-WP, ICI Ag Products) plus 500 mls sterile water for 15min.

Preparation of Agrobacterium

Five to 7 days prior to inoculation, Agrobacterium was streaked from afrozen glycerol stock onto an LB plate (1.5% agar) containing 100 mg/lspectinomycin, 100 mg/l streptomycin, 25 mg/l chloramphenicol, and 50mg/l kanamycin (denoted LBSSCK). Two days before inoculation day, a 10μl loop of Agrobacterium was placed into a tube containg 2 mls of LBSSCKand placed on a rotator overnight at 22-28° C. The day beforeinoculation, the Agrobacterium was subcultured by placing 200 μl in atube containing 2 ml of fresh LBSSCK, which was placed on a rotatorovernight. On the day of inoculation, the Agrobacterium was diluted 1:10with MS liquid medium (Murashige and Skoog, 1962) to an OD₆₆₀ of0.2-0.4.

Explant Inoculation

Sterilized stems were cut into 0.6 cm segments (0.3-1.5 cm segments canbe used), noting their basal orientation. Explants were inoculated forfive minutes in a square Petri plate (100×15 mm) with the 1:10 dilutionof Agrobacterium. Five mls of Agrobacterium solution were added to fivestems by pipetting the Agrobacterium directly on top of the explants.After five minutes, the Agrobacterium solution was aspirated off theexplants. The stem explants were then cultured in the basal-side downorientation for an optimal shoot regeneration response on the co-cultureplates. Co-culture plates (100×15 mm) contained {fraction (1/10)} MSsalts (this can range from about {fraction (1/10)} to full strength;Gibco, 500-1117EH), 1× B5 vitamins (Sigma, G-2519), 0.5 mg/l6-benzylamino-purine (this can range from about 0.1-2 mg/l), 3% sucrose(this can range from about 1-6%), pH 5.7, solidified with 0.9% agar,covered with 2 ml TXD liquid medium (Horsch et al., 1985) onto which an8.5 cm piece of sterile Whatman qualitative grade filter paper wasplaced. Excess Agrobacterium present on the stem explants placed on thefilter paper was blotted off using another piece of sterile 8.5 cmfilter paper. The co-culture plates were placed in clear plastic bagswhich were slit on the sides to permit air exchange, and which wereincubated in a warm room at 25° C. under 24 hours continuous cool whitelight (40 μEn m-²s⁻¹).

Tissue Selection and Regeneration

After two days, the stem explants were moved onto MS medium containing500 mg/l ticarcillin, 50 mg/l cefotaxime, and 1 mg/l6-benzylamino-purine for a three day delay period. Plates were againplaced in slit, clear plastic bags which were placed in the warm room.After a three day delay period, stem explants were moved onto glyphosateselection medium containing MS salts, B5 vitamins, 0.1 mM glyphosate(this can range from about 0.025-0.2 mM), 500 mg/l ticarcillin (this canrange from about 250-750 mg/l), 50 mg/l cefotaxime (this can range fromabout 25-100 mg/l), and 1 mg/l 6-benzylamino-purine (this can range fromabout 0. 1-4 mg/l) for three weeks. After three weeks, the stem explantswere moved onto glyphosate selection medium containing MS salts, B5vitamins, 0.1 mM glyphosate (this can range from about 0.025-0.2 mM),500 mg/l tricarcillin (this can range from about 250-750 mg/l), 50 mg/lcefotaxime (this can range from about 25-100 mg/l), and 1 mg/l6-benzylaminopurine (this can range from about 0.1-4 mg/l), plus 0.5mg/l gibberellic acid A3 (this can range from about 0.1-2 mg/l), whichenhances shoot elongation, for another three week period. After thesesix weeks on glyphosate selection medium, normally developing greenshoots were excised from the stem explants. Shoots (4-5 per plate) wereplaced in rooting medium ({fraction (1/10)}-full strength MS salts,Staba vitamins (Staba, 1969), 3% sucrose (this can range from about1-6%), 500 mg/l ticarcillin (this can range from about 250-750 mg/l), 50mg/l cefotaxime (this can range from about 25-100 mg/l), and 2 mg/lindolebutyric acid (this can range from about 0.5-3 mg/l), pH 5.7,solidified with 0.9% agar. Root development began to occur as early asone week after shoots were placed on rooting medium. At the two weektimepoint, shoots having a large root base were moved into 2½″ potscontaining Metro Mix 350 (Hummert Co., St. Louis, Mo.). Flats werecovered with clear plastic domes (Hummert Co., St. Louis) so the shootscould elongate. Flats containing R₀ plants were placed in a growthchamber under the same conditions as described above for stock plantgrowth. After 34 days, the domes were cracked in order to harden off theplants under the following conditions: Temperature: 20° C. day/15° C.night; Photoperiod: 16 hr light/8 hr dark; Light intensity: 450 μEnm−²s⁻¹; Relative humidity: 70%; Fertilizer: 15-16-17 Peter's Solution(200 ppm nitrogen). Hardened plants were grown for approximately 14weeks under the same conditions, at which time seeds were collected.Cross-pollination was prevented by bagging the plants at bolting time.

This protocol results in transformation efficiencies (defined as thenumber of confirmed transgenics/the number of explants inoculated,expressed as a percentage) as high as 35-40%. This is a significantimprovement over the protocol using kanamycin selection (Fry et al.,1987).

Soybean Transformation

Plant Material

Seeds of soybean variety A3237 were surface sterilized by rinsing themin dilute Tween 20 (polyoxyethylenesorbitan monolaurate) for 30 seconds,followed by rinsing under running tap water for approximately two min.The seeds were then rinsed in 80% ethanol, and then agitated in freshlymade 50% Chlorox (5.25% sodium hypochlorite) containing Tween 20 for 15min. The seeds were then completely rinsed with five rinses of steriledistilled water. They were then placed in a saturated Captan and/orBenylate slurry for 2-30 min to control fungus infestation.

Sterilized seeds were then placed on 0.7% purified agar-solidified B5basal medium (Gamborg et al., 1968) for germination (approximately 15seeds per plate). The Petri dishes were placed in a plastic bag slit onthe sides to permit air exchange, and incubated in a culture room under18-20 hrs light (60 μEn m⁻²s⁻¹), 4-6 hrs dark, at 25° C., for 5-6 days.After this incubation, the germinated seeds were placed in a cold roomor refrigerator (0-10° C.; average temperature of 4° C.) for at least 24hrs prior to explanting.

Preparation of Agrobacterium

Agrobacterium strains to be used for transformation were prepared asfollows. Bacteria were streaked from frozen glycerol stocks onto LBSCKplates containing 1.5% agar-solidified LB medium plus 100 mg/l ofspectinomycin, 25 mg/l of chloramphenicol, and 50 mg/l of kanamycin. Thebacteria can be incubated at room temperature or in an incubator at 27°C. for 24 days. Prior to preparing the Agrobacterium inoculum, a freshplate of Agrobacterium was streaked from the first plate 2-3 days priorto growth on liquid medium. One to two days prior to the inoculation ofsoybean explants, one loop of bacteria was transferred from a freshlystreaked plate into a culture tube containing 2 ml of YEP mediumcontaining 10 g/l peptone, 10 g/l yeast extract, 5 g/l NaCl, 100 mg/lspectinomycin, 25 mg/l chloramphenicol, and 50 mg/l kanamycin. Largervolumes of bacteria can be grown using the same basic formula of oneloop of bacteria per 2 ml of YEP. The tube containing the bacteria inYEP was vortexed to disperse the clump of bacteria, and placed on arotator. For a one day culture, the bacteria can be started at about7:00 a.m.; for a a two day culture, the bacteria can be started later inthe day and allowed to grow overnight. The afternoon prior toinoculating the explants, 4-6 mls (2-3 tubes) of the bacterial culturewere added to 50 mls of AB minimal salts medium (Chilton et al., 1974)containing the same concentrations of spectinomycin, chloramphenicol,and kanamycin as in the LBSCK medium, in sterile 250 ml flasks. Thisculture was grown on a shaker overnight at 28° C. The bacteria werepelleted by centrifugation and the pellet was resuspended to an OD₆₆₀ of0.25-1.0 with the following medium: 1/10 B5 salts (this can range fromabout {fraction (1/10)} to full strength), {fraction (1/10)} B5 vitamins(this can range from about 1110 to full strength), 3% sucrose or glucose(this can range from about 0.5-6% sucrose or glucose), 7.5 μM6-benzyl-aminopurine (this can range from about 2.5-20 μM), 200 μMacetosyringone (this can range from about 50-300 μM), 1 mM galacturonicacid (this can range from about 0.1-2mM), 0.25 mg/l gibberellic acid(GA3) (this can range from 0-0.5 mg/l), and 20 mM MES, pH 5.4 (the pHcan range from about 5.2-6.0).

Explant Inoculation

Explants were prepared by removing the seed coat from the germinatedseedlings and cutting the hypocotyl at approximately 0.5 cm or more fromthe cotyledons (one cm is preferred). The lower portion of the hypocotyland root axis was discarded. The cotyledons and remaining hypocotyl werecompletely split by making an incision down the middle of the hypocotyland then bending the halves apart so that they separated from oneanother. The primary leaves and primary shoot meristem were removed. Theregion of the cotyledon near the axillary bud was wounded multiple times(anywhere from 3-15 times) using a scalpel blade, the score marks beingplaced longitudinally with respect to the embryo axis. The axillary budcan be damaged in the process, but this is not required. Approximately40-80 explants were prepared and added to a single, dry Petri dish.Approximately 10 mls of the bacterial inoculum were added to just coverthe explants. The explants remained in contact with the Agrobacteriumsolution for 30 min. The Agrobacterium solution was then removed fromthe explants which were briefly blotted on sterile Whatman filter paperprior to being placed flat (adaxial) side down onto co-culture plates.Co-culture plates were prepared by adding 4-5 mls of the bacterialdilution medium additionally containing 3% sucrose, 1 mM galacturonicacid, and 200 μM acetosyringone to 1-2 layers of sterile Whatman filterpaper in a 100×15 mm Petri dish. The co-culture medium can contain amixture of 0.5-6% glucose or 0.5-6% sucrose (1-3% of either beingpreferred), with or without 0.1-10 mM galacturonic acid (1 mM beingpreferred), with or without 50-300 μM acetosyringone (100-200 μM beingpreferred). The co-culture medium was solidified with 0.8% washed agar(Sigma, A 8678).

Tissue Selection and Regeneration

The explants were co-cultured with the Agrobacterium in a culture roomat 20-23° C. under an 18-20 hr light/4-6 hr dark photoperiod(co-culturing can be carried out from about 18-26° C.). Co-culturelasted for 2-4 days. After co-culture, the explants were washed in washmedium containing {fraction (1/10)} B5 salts (this can range from about1/10 to full strength), {fraction (1/10)} B5 vitamins (this can rangefrom about {fraction (1/10)} to full strength), 7.5CM6-benzylaminopurine (this can range from about 2.5-20 μM), pH 5.6 (thepH can range from about 5.2-6.0), 500 mg/l ticarcillin (this can rangefrom about 250-750 mg/l), and 100 mg/l cefotaxime (this can range fromabout 25-200 mg/l).

The washed explants were cultured on a culture medium containingB5-basal salts and vitamins, 7.5 μM 6-benzylaminopurine (this can rangefrom about 2.5 μM-20 μM), 500 mg/l ticarcillin (this can range fromabout 250-750 mg/l), 100 mg/l cefotaxime (this can range from about25-200 mg/l), and 0.075-0.1 mM glyphosate (this can range from about0.025-0.4 mM). The plates were sealed with white 3M porous tape andplaced in a culture room or incubator at 24-26° C. under an 18-20 hrlight/66 hr dark cycle at 20-80 μEn m⁻²s⁻¹. Subsequent subcultures weremade every 2-3 weeks.

At two to four weeks, the cultures were transferred to MSB5 medium(Sigma, M 0404 or Gibco, 500-117EH plus Sigma, G2519) or B5 basal mediumplus 1 mg/l zeatin riboside (this can range from about 0-5 mg/l), 0.5mg/l gibberillic acid (GA3) (this can range from about 0-2 mg/l), 0.1mg/l indoleacetic acid (this can range from about 0-1 mg/l), 2.5 μM6-benzylaminopurine (this can range from about 0-5 μM), 500 mg/lticarcillin (this can range from about 250-750 mg/l), 100 mg/lcefotaxime (this can range from about 25-200 mg/l), and 0.075 mMglyphosate (this can range from about 0.025-0.2 mM). Additional B5micronutrients (up to four times the standard concentration of eachmicronutrient alone or in various combinations with the others) and 2gm/l proline (this can range from about 0-2 gm/l) can be added to thismedium.

At the four to six week time point, the petiole/hypocotyl tissue andcotyledons, as well as any dead or dying material, i.e., anynon-regenerating tissues, were removed (such material can generally beremoved between 4-9 weeks). The regenerating cultures were transferredto 0.8% washed agar-solidified elongation medium comprising MSB5 mediumor B5 basal medium plus 1 mg/l zeatin riboside (this can range fromabout 0-5 mg/l), 0.5 mg/l gibberillic acid (this can range from about0-2 mg/l), 0.1 mg/l indoleacetic acid (this can range from about 0-1mg/I), 500 mg/l ticarcillin (this can range from about 250-750 mg/l),100 mg/l cefotaxime (this can range from about 25-200 mg/l), and 0.05 mMglyphosate (this can range from about 0.025-0.2 mM), and again placed ina culture room or incubator at 24-26° C. under an 18-20 hr light/4-6 hrdark cycle at 20-80 μEn m⁻²s⁻¹. Elongation medium can contain about0.25-2 mg/l zeatin riboside, 0.01-1 mg/l indoleacetic acid, and 0.1-5mg/l gibberellic acid (GA3). Cultures were transferred every three weeksto the same medium. Identification of putative A3237 transgenics(elongating, normal appearing shoots) required approximately 8-20 weeks.

Shoots were rooted on 0.7% purified agar-solidifed one-half or fullstrength MSB5 medium or one-half or full strength B5 basal mediumcontaining 500 mg/l ticarcillin (this can range from about 0-500 mg/l),100 mg/l cefotaxime (this can range from about 0-100 mg/l), and 1 mg/lindolebutyric acid (this can range from about 0.1-2 mg/l) ornaphthaleneacetic acid (this can range from about 0.05-2 mg/l), with0-50 mg/l glutamine and 0-50 mg/l asparagine at 24-26° C. under an 18-20hr light/4-6 hr dark cycle for 2-6 weeks. Rooted shoots were placed in2″ pots containing moistened MetroMix 350, and kept enclosed in magentaboxes until acclimatized at 24-26° C. under an 18-20 hr light/4-6 hrdark cycle (20-80 μEn m⁻²s⁻¹). Shoots were hardened off for 3-4 daysafter cracking the lids under the following conditions: Photoperiod:18-20 hrs light/4-6 hrs dark; Light intensity: 20-80 μEn m⁻²s⁻¹;Temperature: 24-26° C. Hardened plants were grown for approximately 3weeks under the following conditions: Photoperiod: 12 hr light/12 hrdark; Light intensity: 450 μEn m⁻²s⁻¹; Relative humidity: 70%;Temperature: 26° C. day/21° C. night. Transformation was confirmed bydetection of expression of the CP4 gene by an ELISA assay. CP4-positiveplants were subsequently grown under the following conditions:Photoperiod: 12 hr light/12 hr dark; Light intensity: 450 μEn m⁻²s⁻¹;Relative humidity: 70%; Temperature: 26° C. day/21° C. night;Fertilizer: 15-16-17 Peter's Solution (200 ppm nitrogen). Plants weregrown for approximately 11 weeks, at which time seed was collected.

Glyphosate Selection

Glyphosate (0.05 mM-0.1 mM) was employed as a selectable marker (Hincheeet al., 1994) for both canola and soybean. Leaves ofglyphosate-resistant canola and soybean transformants (designated R₀generation) were screened for CP4 EPSPS expression by ELISA (Padgette etal., 1995). Seeds from R₀ CP4-positive plants were assayed enzymaticallyfor either the PhbA β-ketothiolase or PhbB reductase, depending upon theplasmid used for transformation (data not shown). PHA synthase was notmeasured enzymatically; however, this gene is carried on the samegenetic insert as the phbB reductase (see FIG. 23), and plantscontaining PhbB activity are likely to contain PhbC activity as well.Soybean and canola seed extracts were prepared by grinding the seeds toa fine powder in liquid N₂, washing with acetone three times, andfiltering through Whatman 3 MM paper. The seed extracts were dried at37° C., one ml of extraction buffer (100 mM KPi, pH7.4, 5 mM MgCl₂, 1 mMEGTA, 5 mM DTT, 10% glycerol) was added, the mixture was vortexed, andthe insoluble fraction was pelleted. The extracts were used for enzymeassays and to detect all PHB biosynthetic enzymes by Western blotanalysis according to Nawrath et al. (1994). Immunoreactive bandscorresponding to each of these enzymes were detected in extracts ofseeds of both plants (data not shown).

Since the CP4 EPSPS gene is genetically linked to the β-ketothiolasegene in pMON25638, and the reductase and PHA synthase genes inpMON25626, glyphosate spray selection (Padgette et al., 1995) wasemployed to identify plants retaining the CP4 EPSPS locus and respectivelinked PHB biosynthetic genes. Plants surviving the glyphosate spraycontain the CP4 EPSPS gene and the linked PHB biosynthetic genes.

Several positive reductase-(pMON25626) and β-ketothiolase-(pMON25638)expressing plants were tested for segregation by spraying glyphosate onapproximately 40 siblings of the R₁ generation (derived byself-pollination of R₀ plants). Any plant that lost the CP4 EPSPS genedue to genetic segregation will die when sprayed with glyphosate. Plantlines that segregated three CP4 positive:one CP4 negative (3:1,indicating the presence of only a single genetic locus) were retainedfor production of homozygous plants for the respective β-ketothiolaseand reductase loci. For each independent line that segregated 3:1,approximately ten R₁ sibling plants per line were retained for seedproduction. Seeds from each of the siblings (designated R₂ generation)were planted, and approximately 40 plants per line were sprayed withglyphosate. Lines in which all R₂ siblings survived the glyphosate spraywere considered to be homozygous for the respective loci.

In order to assemble the entire PHB biosynthetic pathway in a singleplant, homozygous plants derived from transformed canola or soybeancontaining pMON25626 (PhbC and PhbB) were crossed with homozygous plantscontaining pMON25638 (PhbA). Seeds from the crosses (designated F₁) wereassayed for the presence of the β-ketothiolase and reductase to confirmsuccessful crossing (data not shown). At this point in the breedingcycle, the plants are not homozygous for all three genes. The locuscontaining the bketothiolase gene segregates independently from thelocus containing the reductase and synthase genes. The segregation ratioin the F₁ generation would follow a typical Mendelian pattern ofinheritance. Five F₁ progeny seeds were planted, and seeds obtainedtherefrom (designated F₂) were collected and assayed for the presence ofPHB as described below.

Isolation and Characterization of Poly(3-Hydroxybutyrate) fromTransgenic Canola Seeds

40 g of F₂ transgenic canola seeds were crushed in a mechanical benchtopcrusher (Cemotec 1090 Sample Mill, Grinding Disc set to position 1) andthen transferred to cellulose extraction thimbles (Whatman, Singlethickness, 26 mm external diameter×60 mm external length). Extractionwas carried out in these thimbles under refluxing chloroform at 80° C.using a Soxtec Extraction System (Tecator).

After approximately 12 hours of extraction, the extract was collected ina flask, and the chloroform evaporated in a fume-hood under a stream ofair/nitrogen, leaving behind canola oil and an insoluble, gel-like solidresidue. To this mixture, 50 ml of hexanes were added, and the mixturewas stirred for 5 minutes to dissolve the oil. The mixture was thencentrifuged for 5 minutes at 1,700× g, producing a white pellet. Thesupernatant was decanted off, and the pellet redissolved in 10 mlchloroform. Upon stirring this solution with 100 ml hexanes, a whiteprecipitate was obtained. This precipitate was recovered bycentrifugation, washed twice with 30 ml hexanes to remove the residualoil, and dried under a stream of nitrogen for two hours at roomtemperature, and under vacuum for three hours at approximately 40-50° C.

Structural analysis of a 3 mg/ml solution of the precipitate indeuterated-chloroform (99.8% D, Aldrich Chemical Co., USA) by a 300-MHz¹H-NMR performed on a Varian VX-300 spectrometer confirmed it to be purePoly(3-hydroxybutyrate) (P(3HB)) (data not shown).

Molecular weight data were obtained at 35° C. using a Waters MillenniumGPC System and a 410 Differential Refractometer with WatersUltrastyragel Columns of pore sizes 105 Å, 104 Å, 103 Å and 500 Åconnected in series. Chloroform was used as eluent at a flow rate of 1ml/min, and a sample concentration of 6 mg/ml was used. Polystyrenestandards with a low polydispersity (Aldrich Chemical Company, USA) wereused to prepare a calibration curve. The results are shown in Table 13and FIG. 26.

TABLE 13 Production of P(3HB) in Transgenic Canola Seeds Weight ofWeight of % PHB canola PHB (with respect to Mw¹ Mn² Polydispersity seedsobtained seed weight) (Daltons) (Daltons) (Mw/Mn) 40 g 0.2 g 0.5 %686,300 276,800 2.5 ¹Mw: Weight average molecular weight. ²Mn: Numberaverage molecular weight.

Isolation and Characterization of Poly(3-Hydroxtbutyrate) fromTransgenic Soybean Seeds

The extraction procedure described above for canola seeds was alsoemployed for transgenic soybean seeds, using 10 g of F₂ generationcrushed seeds. The results are as summarized in Table 14 and FIG. 27.

TABLE 14 Production of P(3HB) in Transgenic Soybean Seeds Weight ofWeight of % PHB soybean PHB (with respect to Mw¹ Mn² Polydispersityseeds obtained seed weight) (Daltons) (Daltons) (Mw/Mn) 10 g 0.0015 g0.015 % 209,700 99,400 2.1 ¹Mw: Weight average molecular weight. ²Mn:Number average molecular weight.

The data presented in Tables 13 and 14, respectively, confirm thebiosynthesis of P(3HB) in canola seeds at a level of at least 0.5% oftotal seed weight, as well as the biosynthesis of P(3HB) in soybeanseeds at a level of at least 0.015% of the total seed weight, bytransformation and breeding employing plastid-targeted, seed-specificexpression of the A. eutrophus PHB biosynthetic enzymes. These resultsrepresent the first definitive demonstration of the production of PHA inoilseeds of transgenic commercial crops.

EXAMPLE 11 Optimization of P(3HB-co-3HV) Copolymer Production in Plantand Bacterial Cells

The ability to biosynthesize PHAs such as P(3HB-co-3HV) copolymer can beconferred upon heterologous host cells by introduction therein ofappropriate enzyme-encoding nucleic acids in such a manner that theyexpress enzymatically active products. Introduction into bacterial andplant cells of genetic constructs containing chromosomal DNAs, plasmidDNAs, cDNAs, and synthetic DNAs encoding PHA biosynthetic enzymes, invarious combinations with the enzymes discussed above, wherein suchnucleic acids are operably linked to appropriate regulatory regions forexpression depending upon the host cell, enables such cells tobiosynthesize and accumulate PHAs. In bacteria for example, Slater etal. (1988) and Schubert et al. (1988) have demonstrated that E. colitransformed with and expressing the A. eutrophus phbA, phbB, and phbCgenes produces significant levels of PHB. In plants, Poirier et al.(1992) have shown that Arabidopsis expressing these genes produces thePHB homopolymer. Higher levels of PHB were obtained by targeting the Phbenzymes to the leaf plastids (Nawrath et al., 1994).

In view of the ability of heterologous bacterial and plant host cells toproduce PHB polyhydroxyalkanoate upon introduction and expressiontherein of DNAs encoding the appropriate PHB biosynthetic enzymes, it issimilarly expected that P(3HB-co-3HV) copolymer can be produced inbacteria and plants by expressing therein appropriate combinations ofPHA-biosynthetic and other enzymes as discussed above. As the levels ofacetyl-CoA required for copolymer biosynthesis in bacteria and plantcells appear to be non-limiting (Nawrath et. al., 1994), no furthermanipulation of cellular metabolism with respect to this precursor islikely to be necessary. Insuring the presence of sufficient pools ofpropionyl-CoA required for C4/C5 copolymer production may requireintroduction into, and overexpression in, host cells of variouscombinations of wild-type or deregulated aspartate kinase, homoserinedehydrogenase, threonine synthase, wild-type or deregulated threoninedeaminase, α-ketoacid dehydrogenase E1, E2, and E3 subunits, pyruvateoxidase, and acyl-CoA synthetase enzymes.

Thus, DNA encoding the following enzymes can be introduced into andexpressed in plants and bacteria in which P(3HB-co-3HV) copolymer isdesired: an appropriate β-ketothiolase or combination ofβ-ketothiolases, a β-ketoacyl-CoA reductase, and a PHA synthase. Ifnecessary, DNA encoding a wild-type or deregulated threonine deaminasecan also be introduced into and expressed in these organisms. Theβ-ketothiolase can be the BktB β-ketothiolase of A. eutrophus, or onesimilar thereto, that can condense two molecules of acetyl-CoA toproduce acetoacetyl-CoA, and that can also condense acetyl-CoA andpropionyl-CoA to produce β-ketovaleryl-CoA. Alternatively, a combinationof β-ketothiolases can be employed, i.e., a β-ketothiolase capable ofcondensing two molecules of acetyl-CoA to produce acetoacetyl-CoA, and aβ-ketothiolase capable of condensing acetyl-CoA and propionyl-CoA toproduce β-ketovaleryl-CoA. The β-ketoacyl-CoA reductase can be one suchas that encoded by the A. eutrophus phbB gene; the PHA synthase can beone such as that from A. eutrophus as well. A. eutrophus is only one ofmany microorganisms that produce PHAs, and DNA (genomic, plasmid, cDNA,or synthetic DNA) encoding any of the foregoing enzymes can be derivedfrom any other PHA-producing organism containing enzymes having the sameor similar enzymatic activity as the A. eutrophus enzymes, as discussedabove in Example 8 in the section entitled “Other P-Ketothiolases,O-Ketoacyl-CoA Reductases, and PHA Synthases for the Production ofP(3HB-co-3HV) Copolymer.”

As described in Examples 1-8, there are a number of different methods bywhich the levels of α-ketobutyrate, propionyl-CoA, and ultimately3hydroxy-valeryl-CoA which can be utilized in PHA synthesis can beincreased in heterologous bacterial and plant host cells. These aresummarized below.

Overexpression of Threonine Deaminase

The simplest method of increasing the level of α-ketobutyrate in abacterial or plant host cell involves overexpressing a wild-type orderegulated threonine deaminase in such cells. In plants, this enzymecan be expressed cytoplasmically, targeted to the plastids usingappropriate chloroplast transit peptides, or expressed directly withinthese organelles by plastid transformation. These strategies arediscussed in more detail below.

Overexpression of Aspartate Kinase and Threonine Deaminase

Overexpression of a wild-type or deregulated aspartate kinase willresult in an increase in intracellular levels of L-threonine.Deregulated aspartate kinases have been described, for example, in E.coli (Boy et al., 1979; Karchi et al., 1993), C. lactofermentum (Jettenet al., 1995), barley (Bright et al., 1982), maize (Dotson et al., 1990;Hibberd et al., 1980), carrot (Cattoir-Reynaerts et al., 1983), andtobacco (Frankard et al., 1991). Expression of these enzymes in thecytoplasm, targeting of these enzymes to the plastids of plant cells, orexpression of these enzymes in plastids following plastidtransformation, will result in an increase in L-threonine therein.Simultaneous overexpression of a wild-type or deregulated threoninedeaminase in bacterial cells, or in the cytoplasm or plastids of plantcells, will result in conversion of this excess L-threonine toα-ketobutyrate, which will then lead to increased levels ofpropionyl-CoA and then 3-hydroxyvaleryl-CoA, which can be employed inP(3HB-co-3HV) copolymer biosynthesis.

Overexpression of Aspartate Kinase, Homoserine Dehydrogenase, andThreonine Deaminase

An overexpressed homoserine dehydrogenase can be used in conjunctionwith a wild-type or deregulated aspartate kinase to increase further thelevel of threonine in bacterial cells, or in plant cells and plastids byemploying appropriate chloroplast transit peptides or chloroplasttransformation. The excess threonine can then be directed to3-hydroxyvaleryl-CoA synthesis via α-ketobutyrate by an overexpressedwild-type or deregulated threonine deaminase, which can be targeted toor expressed in plastids in plant cells.

Overexpression of Threonine Deaminase, Pyruvate Oxidase, and Acyl-CoASynthetase

α-ketobutyrate levels can be enhanced in bacterial cells and plantplastids (via chloroplast transit peptide targeting or plastidtransformation) by overexpressing a wild-type or deregulated threoninedeaminase. The excess α-ketobutyrate can then be converted to propionateand CO₂ by an overexpressed pyruvate oxidase, for example, E. coli PoxB.Endogenous or overexpressed bacterial or plastid acyl-CoA synthetaseswill then activate the free propionate to produce propionyl-CoA, whichcan then be used by plants expressing other appropriate genes (forexample, isoleucine-deregulated threonine deaminase and BktBβ-ketothiolase) to form the 3-hydroxyvaleryl-CoA monomer employed inP(3HB-co-3HV) copolymer synthesis.

Overexpression of Aspartate Kinase, Homoserine Dehydrogenase, ThreonineDeaminase, Pyruvate Oxidase, and Acyl-CoA Synthetase

A wild-type or deregulated threonine deaminase, aspartate kinase,homoserine dehydrogenase, pyruvate oxidase, and an acyl-CoA synthetase,each having the effect variously described above, can be simultaneouslyoverexpressed in bacterial cells or plant cells and plastids (via theuse of a chloroplast transit peptide or plastid transformation) toprovide increased levels of propionyl-CoA and, eventually,3-hydroxyvaleryl-CoA, which can be incorporated into P(3HB-co-3HV)copolymer in the presence of β-ketothiolase, acetoacetyl-CoA reductase,and PHA synthase.

Overexpression of Aspartate Kinase, Homoserine Dehydrogenase, ThreonineSynthase, Threonine Deaminase, Pyravate Oxidase, and Acyl-CoA Synthetase

In addition to aspartate kinase, homoserine dehydrogenase, threoninedeaminase, pyruvate oxidase, an acyl-CoA synthetase, and threoninesynthase can also be introduced into plant and bacterial cells toprovide increased levels of propionyl-CoA and, eventually,3-hydroxyvaleryl-CoA, which can be incorporated into P(3HB-co-3HV)copolymer in the presence of β-ketothiolase, acetoacetyl-CoA reductase,and PHA synthase.

Overexpression of a Branched-Chain α-Ketoacid Dehydrogenase Complex E1Subunit

As discussed in Example 6, pyruvate dehydrogenase complex catalyzes theoxidative decarboxylation of α-ketobutyrate to form propionyl-CoA.Pyruvate dehydrogenase complex is a multienzyme complex containing threeactivities: a pyruvate decarboxylase (E1); a dihydrolipoyltransacetylase (E2); and a dihydrolipoyl dehydrogenase (E3).

Overexpression of a branched-chain α-ketoacid dehydrogenase E1 subunithaving improved binding and decarboxylating activity with α-ketobutyrateas compared to the naturally occurring E1 subunit may effectivelycompete with the endogenous bacterial or plant E1 subunit. Thisoverexpressed E1 subunit will combine with the endogenous pyruvatedehydrogenase E2E3 subcomplex to create a functional hybrid complexcapable of turning over α-ketobutyrate to propionyl-CoA, which can thenbe metabolized to 3-hydroxyvaleryl-CoA for incorporation intoP(3HB-co-3HV) copolymer.

Alternatively, both the E1 and E2 components of a branched-chainα-ketoacid dehydrogenase complex that has significant activity withα-ketobutyrate can be overexpressed. Overexpression of the dihydrolipoyldehydrogenase E3 subunit may not be necessary as this subunit is commonto all α-ketoacid dehydrogenase complexes. However, if the endogenous E3levels are not sufficient for E1 E2 of endogenous PDC and theoverexpressed E1 E2 of a branched-chain α-ketoacid dehydrogenasecomplex, then it too can be overexpressed.

Overexpression of an α-ketoacid dehydrogenase complex E1 subunit havinggreater binding and decarboxylating activity with α-ketobutyrate thanthe naturally occurring E1 subunit in bacterial cells or plant plastids,or overexpression of both E1 and E2 components as discussed above, canbe employed in conjunction with any of the other methods discussedherein as a means for increasing the amounts of propionyl-CoA andsubsequently 3-hydroxyvaleryl-CoA available for P(3HB-co-3HV) copolymersynthesis.

Production of Transformed Bacteria and Transgenic Plants Capable ofProducing P(3HB-co-3HV) Copolymer

PHA synthesis in bacteria and plants can be optimized in accordance withthe present invention by expressing DNAs encoding β-ketothiolase,β-acyl-CoA reductase, and PHA synthase in conjunction with variouscombinations of precursor-producing enzymes, as discussed in theforegoing Examples. Methods therefor are discussed below.

Bacterial Vectors

Methods for incorporating all the genes discussed herein intotransformation/expression vector constructs and introducing theseconstructs into bacterial and plant host cells to produce PHAs in suchcells are well known in the art. Poirier et al. (1995) have recentlyprovided an extensive review of progess in this area. In general, suchvector constructs comprise assemblies of DNA fragments operativelylinked in a functional manner such that they drive the expression of thestructural DNA sequences contained therein. These vector constructsusually contain a promoter that functions in the selected host cell,along with any other necessary regulatory regions such as ribosomebinding sites, transcription terminators, 3′ non-translatedpolyadenylation signals, etc., linked together in an operable manner, aswell as selectable markers (Sambrook et al., 1989; Ausubel et al. 1989).Vectors for bacterial cloning have been reviewed in Rodriguez et al.(1988).

Bacterial Transformation

Such vectors can be introduced into bacterial cells by calciumchloride/heat shock treatment or electroporation. Transformed host cellscan subsequently be selected on selective media, cultured in anappropriate medium for a time and under conditions conducive to theproduction of PHA, and the PHA can then be recovered. Representativemethods have been described by Slater et al. (1988); Slater et al.(1992); Zhang et al. (1994); and Kidwell et al. (1995).

Bacterial and Other Host Cells

Useful host organisms for PHA polymer production include Actinomycetes(e.g., Streptomyces sp. and Nocardia sp.); bacteria (e.g., Alcaligenes(e.g., A. eutrophus), Bacillus cereus, B. subtilis, B. licheniformis, B.megaterium, Escherichia coli, Klebsiella (e.g., K. aerogenes and K.oxytoca), Lactobacillus, Methylomonas, Pseudomonas (e.g., P. putida andP. fluorescens); fungi (e.g., Aspergillus, Cephalosporium, andPenicillium); and yeast (e.g., Saccharomyces, Rhodotorula, Candida,Hansenula, and Pichia).

Organisms capable of overproducing threonine either naturally, viachemical or physical mutagenesis, or via recombinant DNA methodology(Jetten & Sinskey, 1995), can be used in the present invention for theproduction of P(3HB-co-3HV) copolymer. DNAs selected from the groupconsisting of DNA encoding a wild-type or deregulated threoninedeaminase, a β-ketothiolase capable of condensing two molecules ofacetyl-CoA to produce acetoacetyl-CoA, a β-ketothiolase capable ofcondensing acetyl-CoA and propionyl-CoA to form β-ketovaleryl-CoA, e.g.,BktB, β-ketoacyl-CoA reductase capable of converting a β-ketoacyl-CoA toits corresponding β-hydroxyacyl-CoA, e.g., PhbB, and a PHA synthasecapable of producing P(3HB-co-3HV) copolymer from theβ-hydroxyacyl-CoAs, e.g., PhbC, or combinations thereof, can beintroduced into these organisms in order to impart P(3HB-co-3HV)copolymer production capability thereto.

Organisms that are capable of producing propionate or propionyl-CoA andother odd-chain substrates from simple precursors such as glucose,lactate, etc., are also useful for producing P(3HB-co-3HV) copolymer.For example, DNAs encoding a β-ketothiolase capable of condensing twomolecules of acetyl-CoA to produce acetoacetyl-CoA, a β-ketothiolasecapable of condensing acetyl-CoA and propionyl-CoA to formβ-ketovaleryl-CoA, e.g., BktB, a β-ketoacyl-CoA reductase capable ofconverting a β-ketoacyl-CoA to its corresponding β-hydroxyacyl-CoA,e.g., PhbB, and a PHA synthase capable of producing P(3HB-co-3HV)copolymer from the β-hydroxyacyl-CoAs, e.g., PhbC, or combinationsthereof, can be introduced into these organisms. Examples of suchorganisms include members of the genus Clostridium, such as Clostridiumarcticum, Clostridium novyi, Clostridium propionicum, as well asMegasphaera elsdenii.

In addition, organisms that are capable of producing butyrate orbutyryl-CoA from simple precursors are useful for producing C4/C6copolymer, i.e., P(3HB-co-3HC) copolymer. For example, DNAs encoding aβ-ketothiolase capable of condensing two molecules of acetyl-CoA toproduce acetoacetyl-CoA, a β-ketothiolase capable of condensingacetyl-CoA and butyryl-CoA to form β-ketocaproyl-CoA, e.g., BktB, aβ-ketoacyl-CoA reductase capable of converting a β-ketoacyl-CoA to itscorresponding β-hydroxyacyl-CoA, e.g., PhbB, and a PHA synthase capableof producing P(3HB-co-3HC) copolymer from the β-hydroxyacyl-CoA, e.g.,Nocardia corallina PHA synthase, or combinations thereof, can beintroduced into these organisms. Examples of such organisms includespecies of the genera Butyrivibrio, Clostridium (e.g., Clostridiumpopuleti), Eubacterium, and Fusarium (Patel and Agnew, 1988).

Plant Vectors

In plants, transformation vectors capable of introducing encoding DNAsinvolved in PHA biosynthesis are easily designed, and generally containone or more DNA coding sequences of interest under the transcriptionalcontrol of 5′ and 3′ regulatory sequences. Such vectors generallycomprise, operatively linked in sequence in the 5′ to 3′ direction, apromoter sequence that directs the transcription of a downstreamheterologous structural DNA in a plant; optionally, a 5′ non-translatedleader sequence; a nucleotide sequence that encodes a protein ofinterest; and a 3′ non-translated region that encodes a polyadenylationsignal which functions in plant cells to cause the termination oftranscription and the addition of polyadenylate nucleotides to the 3′end of the mRNA encoding said protein. Plant transformation vectors alsogenerally contain a selectable marker. Typical 5′-3′ regulatorysequences include a transcription initiation start site, a ribosomebinding site, an RNA processing signal, a transcription terminationsite, and/or a polyadenylation signal. Vectors for plant transformationhave been reviewed in Rodriguez et al. (1988), Glick et al. (1993), andCroy (1993).

Plant Promoters

Plant promoter sequences can be constitutive or inducible,environmentally- or developmentally-regulated, or cell- ortissue-specific. Often-used constitutive promoters include the CaMV 35Spromoter (Odell et al., 1985), the enhanced CaMV 35S promoter, theFigwort Mosaic Virus (FMV) promoter (Richins et al., 1987), themannopine synthase (mas) promoter, the nopaline synthase (nos) promoter,and the octopine synthase (ocs) promoter. Useful inducible promotersinclude heat-shock promoters (Ou-Lee et al., 1986; Ainley et al., 1990),a nitrate-inducible promoter derived from the spinach nitrite reductasegene (Back et al., 1991), hormone-inducible promoters(Yamaguchi-Shinozaki et al., 1990; Kares et al., 1990), andlight-inducible promoters associated with the small subunit of RuBPcarboxylase and LHCP gene families (Kuhlemeier et al., 1989; Feinbaum etal., 1991; Weisshaar et al., 1991; Lam and Chua, 1990; Castresana etal., 1988; Schulze-Lefert et al., 1989). Examples of usefultissue-specific, developmentally-regulated promoters include theβ-conglycinin 7S promoter (Doyle et al., 1986; Slighton and Beachy,1987), and seed-specific promoters (Knutzon et al., 1992; Bustos et al.,1991; Lam and Chua, 1991; Stayton et al., 1991). Plant functionalpromoters useful for preferential expression in seed plastids includethose from plant storage protein genes and from genes involved in fattyacid biosynthesis in oilseeds. Examples of such promoters include the 5′regulatory regions from such genes as napin (Kridl et al., 1991),phaseolin, zein, soybean trypsin inhibitor, ACP, stearoyl-ACPdesaturase, and oleosin. Seed-specific gene regulation is discussed inEP 0 255 378. Promoter hybrids can also be constructed to enhancetranscriptional activity (Hoffman, U.S. Pat. No. 5,106,739), or tocombine desired transcriptional activity and tissue specificity.

A factor to be considered in the choice of promoters is the timing ofavailability of the necessary substrates during expression of the PHAbiosynthetic enzymes. For example, if P(3HB-co-3HV) copolymer isproduced in seeds from threonine, the timing of threonine biosynthesisand the amount of free threonine are important considerations. Karchi etal. (1994) have reported that threonine biosynthesis occurs rather latein seed development, similar to the timing of seed storage proteinaccumulation. For example, if enzymes involved in P(3HB-co-3HV)copolymer biosynthesis are expressed from the 7S seed-specific promoter,the timing of expression thereof will be concurrent with threonineaccumulation.

Plant Transformation and Regeneration

A variety of different methods can be employed to introduce such vectorsinto plant protoplasts, cells, callus tissue, leaf discs, meristems,etc., to generate transgenic plants, including Agrobacterium-mediatedtransformation, particle gun delivery, microinjection, electroporation,polyethylene glycolmediated protoplast transformation, liposome-mediatedtransformation, etc. (reviewed in Potrykus, 1991). In general,transgenic plants comprising cells containing and expressing DNAsencoding enzymes facilitating PHA biosynthesis can be produced bytransforming plant cells with a DNA construct as described above via anyof the foregoing methods; selecting plant cells that have beentransformed on a selective medium; regenerating plant cells that havebeen transformed to produce differentiated plants; and selecting atransformed plant which expresses the enzyme-encoding nucleotidesequence.

Constitutive overexpression of, for example, a deregulated threoninedeaminase employing the e35S or FMV promoter might potentially starveplants of certain amino acids, especially those of the aspartate family.If such starvation occurs, the negative effects may be avoided bysupplementing the growth and cultivation media employed in thetransformation and regeneration procedures with appropriate amino acids.By supplementing the transformation/regeneration media with aspartatefamily amino acids (aspartate, threonine, lysine, and methionine), theuptake of these amino acids into the plant can reduce any potentialstarvation effect caused by an overexpressed threonine deaminase.Supplementation of the media with such amino acids might thereby preventany negative selection, and therefore any adverse effect ontransformation frequency, due to the overexpression of a deregulatedthreonine deaminase in the transformed plant.

The encoding DNAs can be introduced either in a single transformationevent (all necessary DNAs present on the same vector), aco-transformation event (all necessary DNAs present on separate vectorsthat are introduced into plants or plant cells simultaneously), or byindependent transformation events (all necessary DNAs present onseparate vectors that are introduced into plants or plant cellsindependently). Traditional breeding methods can subsequently be used toincorporate the entire pathway into a single plant. Successfulproduction of the PHA polyhydroxybutyrate in is cells of Arabidopsis hasbeen demonstrated by Poirier et al. (1992), and in plastids thereof byNawrath et al. (1994).

Specific methods for transforming a wide variety of dicots and obtainingtransgenic plants are well documented in the literature (Gasser andFraley, 1989; Fisk and Dandekar, 1993; Christou, 1994; and thereferences cited therein).

Successful transformation and plant regeneration have been achieved inthe monocots as follows: asparagus (Asparagus officinalis; Bytebier etal. 1987); barley (Hordeum vulgarae; Wan and Lemaux 1994); maize (Zeamays; Rhodes et al., 1988; Gordon-Kamm et al., 1990; Fromm et al., 1990;Koziel et al., 1993); oats (Avena saliva; Somers et al., 1992);orchardgrass (Dactylis glomerata; Horn et al., 1988); rice (Oryzasativa, including indica and japonica varieties; Toriyama et al., 1988;Zhang et al., 1988; Luo and Wu 1988; Zhang and Wu 1988; Christou et al.,1991); rye (Secale cereale; De la Pena et al., 1987); sorghum (Sorghumbicolor; Cassas et al. 1993); sugar carve (Saccharum spp.; Bower andBirch 1992); tall fescue (Festuca arundinacea; Wang et al. 1992);turfgrass (Agrostis palustris; Zhong et al., 1993); wheat (Triticumaestivum; Vasil et al. 1992; Weeks et al. 1993; Becker et al. 1994), andalfalfa (Masoud, S. A. et al.1996).

Host Plants

Particularly useful plants for PHA copolymer production include thosethat produce carbon substrates which can be employed for PHAbiosynthesis, including tobacco, wheat, potato, Arabidopsis, and highoil seed plants such as corn, soybean, canola, oil seed rape, sunflower,flax, peanut, sugarcane, switchgrass, and alfalfa. Polymers that can beproduced in this manner include copolymers incorporating both shortchain length and medium chain length monomers, such as P(3HB-co-3HV)copolymer.

If the host plant of choice does not produce the requisite fatty acidsubstrates in sufficient quantities, it can be modified, for example bymutagenesis or genetic transformation, to block or modulate the glycerolester and fatty acid biosynthesis or degradation pathways so that itaccumulates the appropriate substrates for PHA production.

P(3HB-co-3HV) Copolymer Composition

The P(3HB-co-3HV) copolymers of the present invention can comprise about75-99% 3HB and about 1-25% 3HV based on the total weight of the polymer.More preferably, P(3HB-co-3HV) copolymers of the present inventioncomprise about 85-99% 3HB and about 1-15% 3HV. Even more preferably,such copolymers comprise about 90-99% 3HB and about 1-10% 3HV.P(3HB-co-3HV) copolymers comprising about 4%, about 8%, and about 12%3HV on a weight basis possess properties that have made themcommercially attractive for particular applications. One skilled in theart can modify P(3HB-co-3HV) copolymers of the present invention byphysical or chemical means to produce copolymer derivatives havingdesirable properties different from those of the plant- ormicrobially-produced copolymer.

Optimization of P(3HB-co-3HV) copolymer production by the methodsdiscussed herein is expected to result in yields of copolymer in therange of from at least about 1% to at least about 20% of the freshweight of the plant tissue, organ, or structure in which it is produced.In bacteria, yields in the range of from at least about 1% to at leastabout 90% of cell dry weight is expected.

Plastid Targeting of Expressed Enzymes for PHA Biosynthesis

PHA polymer can be produced in plants either by expression of theappropriate enzymes in the cytoplasm (Poirier et al., 1992) by themethods described above, or more preferably, in plastids, where higherlevels of PHA production can be achieved (Nawrath et al., 1994). Asdemonstrated by the latter group, targeting of β-ketothiolase,acetoacetyl-CoA reductase, and PHB synthase to plastids of Arabidopsisthaliana results in the accumulation of high levels of PHB in theplastids without any readily apparent deleterious effects on plantgrowth and seed production. As branched-chain amino acid biosynthesisoccurs in plant plastids (Bryan, 1980; Galili, 1995), overexpressiontherein of plastid-targeted enzymes, including a deregulated form ofthreonine deaminase, is expected to facilitate the production ofelevated levels of α-ketobutyrate and propionyl-CoA. The latter can becondensed with acetyl-CoA by β-ketothiolase to form 3-ketovaleryl-CoA,which can then be further metabolized by a β-keto-acyl-CoA reductase to3-hydroxyvaleryl-CoA, the precursor of the C5 subunit of P(3HB-co-3HV)copolymer. As there is a high carbon flux through acetyl-CoA inplastids, especially in seeds of oil-accumulating plants such as oilseedrape (Brassica napus), canola (Brassica rapa, Brassica campestris,Brassica carinata, and Brassica juncea), soybean (Glycine max), flax(Linum usitatissimum), and sunflower (Helianthus annuus) for example,targeting of the gene products of desired encoding DNAs to leucoplastsof seeds, or transformation of seed leucoplasts and expression thereinof these encoding DNAs, are attractive strategies for achieving highlevels of PHA biosynthesis in plants.

All of the enzymes discussed herein can be modified for plastidtargeting by employing plant cell nuclear transformation constructswherein DNA coding sequences of interest are fused to any of theavailable transit peptide sequences capable of facilitating transport ofthe encoded enzymes into plant plastids (partially summarized in vonHeijne et al., 1991), and driving expression by employing an appropriatepromoter. The sequences that encode a transit peptide region can beobtained, for example, from plant nuclear-encoded plastid proteins, suchas the small subunit (SSU) of ribulose bisphosphate carboxylase, plantfatty acid biosynthesis related genes including acyl carrier protein(ACP), stearoyl-ACP desaturase, β-ketoacyl-ACP synthase and acyl-ACPthioesterase, or LHCPII genes. The encoding sequence for a transitpeptide effective in transport to plastids can include all or a portionof the encoding sequence for a particular transit peptide, and may alsocontain portions of the mature protein encoding sequence associated witha particular transit peptide. Numerous examples of transit peptides thatcan be used to deliver target proteins into plastids exist, and theparticular transit peptide encoding sequences useful in the presentinvention are not critical as long as delivery into a plastid isobtained. Proteolytic processing within the plastid then produces themature enzyme. This technique has proven successful not only withenzymes involved in PHA synthesis (Nawrath et al., 1994), but also withneomycin phosphotransferase II (NPT-II) and CP4 EPSPS (Padgette et al.,1995), for example.

Of particular interest are transit peptide sequences derived fromenzymes known to be imported into the leucoplasts of seeds. Examples ofenzymes containing useful transit peptides include those related tolipid biosynthesis (e.g., subunits of the plastid-targeted dicotacetyl-CoA carboxylase, biotin carboxylase, biotin carboxyl carrierprotein, α-carboxy-transferase, plastid-targeted monocot multifunctionalacetyl-CoA carboxylase (Mr, 220,000); plastidic subunits of the fattyacid synthase complex (e.g., acyl carrier protein (ACP), malonyl-ACPsynthase, KASI, KASII, KASIII, etc.); steroyl-ACP desaturase;thioesterases (specific for short, medium, and long chain acyl ACP);plastid-targeted acyl transferases (e.g., glycerol-3-phosphate: acyltransferase); enzymes involved in the biosynthesis of aspartate familyamino acids; phytoene synthase; gibberellic acid biosynthesis (e.g.,ent-kaurene synthases 1 and 2); sterol biosynthesis (e.g., hydroxymethyl glutaryl-coA reductase); and carotenoid biosynthesis (e.g.,lycopene synthase).

Exact translational fusions to the transit peptide of interest may notbe optimal for protein import into the plastid. By creatingtranslational fusions of any of the enzymes discussed herein to theprecursor form of a naturally imported protein or C-terminal deletionsthereof, one would expect that such translational fusions would aid inthe uptake of the engineered precursor protein into the plastid. Forexample, Nawrath et al., (1994) used a similar approach to create thevectors employed to introduce the PHB biosynthesis genes of A. eutrophusinto Arabidopsis.

It is therefore fully expected that targeting of the enzymes discussedin Examples 1-8 to leaf chloroplasts or seed plastids such asleucoplasts by fusing transit peptide gene sequences thereto willfurther enhance in vivo conditions for the biosynthesis of PHAs inplants.

Plastid Transformation for Expression of Enzymes Involved in PHABiosynthesis

Alternatively, enzymes facilitating the biosynthesis of metabolites suchas threonine, α-ketobutyrate, propionyl-CoA, 3-ketovaleryl-CoA,3-hydroxy-valeryl-CoA, and PHAs discussed herein can be expressed insitu in plastids by direct transformation of these organelles withappropriate recombinant expression constructs. Constructs and methodsfor stably transforming plastids of higher plants are well known in theart (Svab et al., 1990; Svab et al., 1993; Staub et al., 1993; Maliga etal., U.S. Pat. No. 5,451,513; PCT International Publications WO95/16783, WO 95/24492, and WO 95/24493). These methods generally rely onparticle gun delivery of DNA containing a selectable marker in additionto introduced DNA sequences for expression, and targeting of the DNA tothe plastid genome through homologous recombination. Transformation of awide variety of different monocots and dicots by particle gunbombardment is routine in the art (Hinchee et al., 1994; Walden andWingender, 1995).

DNA constructs for plastid transformation generally comprise a targetingsegement comprising flanking DNA sequences substantially homologous to apredetermined sequence of a plastid genome, which targeting segmentenables insertion of DNA coding sequences of interest into the plastidgenome by homologous recombination with said predetermined sequence; aselectable marker sequence, such as a sequence encoding a form ofplastid 16S ribosomal RNA that is resistant to spectinomycin orstreptomycin, or that encodes a protein which inactivates spectinomycinor streptomycin (such as the aadA gene), disposed within said targetingsegment, wherein said selectable marker sequence confers a selectablephenotype upon plant cells, substantially all the plastids of which havebeen transformed with said DNA construct; and one or more DNA codingsequences of interest disposed within said targeting segment relative tosaid selectable marker sequence so as not to interfere with conferringof said selectable phenotype. In addition, plastid expression constructsalso generally include a plastid promoter region and a transcriptiontermination region capable of terminating transcription in a plantplastid, wherein said regions are operatively linked to the DNA codingsequences of interest.

A further refinement in chloroplast transformation/expression technologythat facilitates control over the timing and tissue pattern ofexpression of introduced DNA coding sequences in plant plastid genomeshas been described in PCT International Publication WO 95/16783. Thismethod involves the introduction into plant cells of constructs fornuclear transformation that provide for the expression of a viral singlesubunit RNA polymerase and targeting of this polymerase into theplastids via fusion to a plastid transit peptide. Transformation ofplastids with DNA constructs comprising a viral single subunit RNApolymerase-specific promoter specific to the RNA polymerase expressedfrom the nuclear expression constructs operably linked to DNA codingsequences of interest permits control of the plastid expressionconstructs in a tissue and/or developmental specific manner in plantscomprising both the nuclear polymerase construct and the plastidexpression constructs. Expression of the nuclear RNA polymerase codingsequence can be placed under the control of either a constitutivepromoter, or a tissue- or developmental stage-specific promoter, therebyextending this control to the plastid expression construct responsive tothe plastid-targeted, nuclear-encoded viral RNA polymerase. Theintroduced DNA coding sequence can be a single encoding region, or maycontain a number of consecutive encoding sequences to be expressed as anengineered or synthetic operon. The latter is especially attractivewhere, as in the present invention, it is desired to introduce multigenebiochemical pathways into plastids. This approach is not practical usingstandard nuclear transformation techniques since each gene introducedtherein must be engineered as a monocistron, including an encodedtransit peptide and appropriate promoter and terminator signals.Individual gene expression levels may vary widely among differentcistrons, thereby possibly adversely affecting the overall biosyntheticprocess. This can be avoided by the chloroplast transformation approach.

Biosynthesis of higher poly(3-hydroxyalkanoate) polymers in plants

Biosynthesis of higher poly(3-hydroxyalkanoate) polymers in plants frommedium chain length monomers (C6-C12) are possible based on informationfrom the genetics and biochemistry of PHA formation in bacteria,combined with the knowledge of available plant metabolic intermediates.Examples of such pathway intermediates are those that are present duringfatty acid biosynthesis and degradation, as well as intermediates formedthrough the biological processing of carbohydrates and amino acids. Thisknowledge for 3-hydroxyalkanoate formation in plants is well known inthe art. For a general reference on monomers including C₄, C₆, C₈, . . ., C₂₀,C₂₂, see van der Leij and Witholt (1995). Plants engineered toproduce the corresponding monomers could be used in the inventiveprocesses disclosed herein to afford a wide array of homo- andco-polymeric materials.

Biosynthesis of 4-hydroxybutyrate containing polymers in plants

Poly(4-hydroxybutyrate) and copolymers of 4-hydroxybutyrate and otherhydroxyalkanoates such as 3-hydroxybutyrate, 3-hydroxyvalerate, and3-hydroxyhexanoate may be biosynthesized in plants capable of producing4-hydroxybutyryl-CoA. Polymers containing 4-hydroxybutyrate aredesirable due to their improved material properties, e.g. increasedtensile strength, when compared to other short chainpolyhydroxyalkanoates. The substrate 4-hydroxybutyryl-CoA may bebiosynthesized in plants by, for example, the Clostridium kluyveripathway.

The genes involved in the conversion of succinate to 4-hydroxybutyratein Clostridium kluyveri have been fully characterized (Söhling 1996).Succinate is first converted to succinyl-CoA by succinyl-CoA:acetyl-CoACoA transferase. Next, succinyl-CoA is transformed to succinic acidsemialdehyde by succinate semialdehyde dehydrogenase. The enzyme4-hydroxybutyrate dehydrogenase acts to convert succinic acidsemialdehyde to 4-hydroxybutyric acid, which is subsequently convertedto 4-hydroxybutyryl-CoA by 4-hydroxybutyryl-CoA:acetyl-CoA CoAtransferase. Transformation of a plant containing Alcaligenes eutrophus3-polyhydroxybutyrate synthesis genes with the Clostridium kluyveri4-hydroxybutyrate synthesis genes would afford a plant capable ofproducing the 4-hydroxybutyrate containing copolymers.

The available 4-hydroxybutyryl-CoA may be incorporated intopolyhydroxyalkanoate polymers by a PHA synthase.

Control of gone expression in plants

In the present invention it will likely be useful to control the levelof one or more of the PHA enzymes to achieve the desired molecularweight (MW), molecular weight distribution or polydispersity (PDI) andPHA levels in recombinant plants or bacteria. There are several methodsof regulating the expression of one or more enzymes in plants. Forexample, if high level protein expression is desired, one could choose asuitable promoter that has high levels of transcriptional activity, forexample a plant viral promoter such as the 35S promoter (Odell et. al1985.). High level transcription activity of promoters can also be foundthat express in certain tissue types or during certain stages of plantdevelopment for example seed storage protein promoters such as 7S (Doyleet. al. 1986). Transcriptional activity can also be modulated byoverexpression of transcription factors that regulate promoters, oralternatively by removing a repressor that blocks transcription of apromoter.

Other methods for obtaining high level gene expression involve the useof plant viral vectors to deliver one or more genes of interest (Donsonet. al., (1991); Champman et. al., (1992)). Viral RNA expression vectorshave been developed to produce significant quantities of foreignproteins in plants. It would also be possible to use two different RNAvirus vectors that can co-infect the same cells of a plant (i.e. potatovirus Y (PVY) and potato virus X (PVX)). The use of two different viralRNA vectors could be used as a control point for modulating theexpression levels of one or more of the specific protein of interest.For instance, if it is preferred to have a high level production of oneprotein, but low levels of another, then suitable viral RNA vectors canbe constructed to achieve the desired expression levels of the proteins.These desired levels of proteins will be dictated by the relativetranscriptional activity of the respective viral RNA vectors.

Another methods for achieving high level expression and translation ofproteins is to optimize the amino acid codons specific to the targetorganism. By reconstructing the gene encoding the protein of interestwith preferred amino acid codons typical of the target host organism,one can increase the level of translation of the protein of interest inplants. There are also other methods available for increasing thetranslatability of genes in plants including the use of introns and 5′untranslated leader sequences.

Organelle transformation, such as chloroplast (Boynton et. al., 1988;Svab et. al., 1990) and mitochondria transformation (Johnston et. al.1988), has achieved high levels of expression (McBride et. al., 1995) ofengineered proteins in plants since there are hundreds of copies of thechloroplast genome per cell thst would includey our engineered gene.This amplification of the number of copies of the gene, and the use ofstrong chloroplast promoters (i.e. prm) usually results in hightranscription and translation of the recombinant protein expression(McBride et. al., 1995). It is also possible to introduce several genesinto the chloroplast genome as separate transcriptional units or as atypical bacterial operon (Staub and Maliga, 1995). In addition,inserting genes into the inverted repeat regions of the chloroplastgenome results in the production of two copies of the introduced geneper chromosome, thus amplifying the expression even more.

In most cases, high level protein expression is desirable in thetransgenic plants. However, there may be circumstances that high levelprotein expression is not desirable for every protein. For example, toachieve optimal PHA biosynthesis of desirable molecular weight andpolydispersity it may be necessary to control the levels of the PHAproduction proteins. For instance, having high level protein productionof the β-ketothiolase (i.e. PhbA or BktB) and the D-reductase (i. e.PhbB) and low level expression of the PHA synthase (i. e. PhbC) mightresult in the production of high molecular weight PHA of the desiredpolydispersity (Jun Sim et. al., 1997)

For example, one way to achieve high level expression of β-ketothiolaseand D-reductase and low level expression of the PHA synthase would be touse strong promoters for the β-ketothiolase and D-reductase and a weakerpromoter for the PHA synthase. It would also be possible to express theβ-ketothiolase and D-reductase from a promoter that expresses early inseed development and express the PHA synthase from a promoter later inseed development. This might allow the metabolite required by the PHAsynthase to build up in the plant cell and be immediately available forits use in PHA biosynthesis.

Another example would be to introduce the β-ketothiolase and D-reductasein the chloroplast genome and the PHA synthase in the nuclear genome.This would also achieve high expression of the β-ketothiolase andD-reductase and lower expression of the PHA synthase. Another strategywould be to insert the β-ketothiolase and D-reductase into the invertedrepeat region of the chloroplast genome and the PHA synthase into asingle copy region of the chloroplast genome. This would result inhigher production of the β-ketothiolase and D-reductase and lowerproduction of the PHA synthase.

Another method to increase expression of the β-ketothiolase andD-reductase would be to cross a homozygous plant line containing theβ-ketothiolase, D-reductase, and PHA synthase with another homozygousplant line containing just the β-ketothiolase and D-reductase. Thiswould result in two copies of the β-ketothiolase and D-reductase and onecopy of the PHA synthase. This could also be achieved by genereatingnuclear transformants of PHA synthase containing one copy of the gene(this can be determined by Southern blot analysis) and retransformingthe plant by particle gun bombardment with expression plasmidscontaining β-ketothiolase and D-reductase and selecting lines containingmultiple copies of the β-ketothiolase and/or D-reductase.

Finally, one could also alter the amino acid composition of the PHAsynthase to be non-optimal for translation in the target plant orbacteria. This would result in poor translation of the protein and lowerproduction levels of the PHA synthase protein. Alternatively, one couldengineer in the PHA synthase protein signals that target the protein forrapid protein degredation (i.e. ubiquitin dependent degredation). Thiswould also prevent the PHA synthase from accumulating to high levels inthe plant.

Production of Transgenic Plants Comprising Genes for PHA Biosynthesis

Plant transformation vectors capable of delivering DNAs (genomic DNAs,plasmid DNAs, cDNAs, or synthetic DNAs) encoding PHA biosyntheticenzymes and other enzymes for optimizing substrate pools for PHAbiosynthesis as discussed above in Examples 1-8 can be easily designed.Various strategies can be employed to introduce these encoding DNAs toproduce transgenic plants capable of biosynthesizing high levels ofPHAs, including:

1. Transforming individual plants with an encoding DNA of interest. Twoor more transgenic plants, each containing one of these DNAs, can thenbe grown and cross-pollinated so as to produce hybrid plants containingthe two DNAs. The hybrid can then be crossed with the remainingtransgenic plants in order to obtain a hybrid plant containing all DNAsof interest within its genome.

2. Sequentially transforming plants with plasmids containing each of theencoding DNAs of interest, respectively.

3. Simultaneously cotransforming plants with plasmids containing each ofthe encoding DNAs, respectively.

4. Transforming plants with a single plasmid containing two or moreencoding DNAs of interest.

5. Transforming plants by a combination of any of the foregoingtechniques in order to obtain a plant that expresses a desiredcombination of encoding DNAs of interest.

Traditional breeding of transformed plants produced according to any oneof the foregoing methods by successive rounds of crossing can then becarried out to incorporate all the desired encoding DNAs in a singlehomozygous plant line (Nawrath et al., 1994; PCT InternationalPublication WO 93/02187). Similar strategies can be employed to producebacterial host cells engineered for optimal PHA production.

In methods 2 and 3, the use of vectors containing different selectablemarker genes to facilitate selection of plants containing two or moredifferent encoding DNAs is advantageous. Examples of useful selectablemarker genes include those conferring resistance to kanamycin,hygromycin, sulphonamides, glyphosate, bialaphos, and phosphinothricin.

Stability of Transgene Expression

As several overexpressed enzymes may be required to produce optimallevels of substrates for copolymer formation, the phenomenon ofco-suppression may influence transgene expression in transformed plants.Several strategies can be employed to avoid this potential problem(Finnegan and McElroy, 1994).

One commonly employed approach is to select and/or screen for transgenicplants that contain a single intact copy of the transgene or otherencoding DNA (Assaad et al., 1993; Vaucheret, 1993; McElroy andBrettell, 1994). Agrobacterium-mediated transformation technologies arepreferred in this regard.

Inclusion of nuclear scaffold or matrix attachment regions (MAR)flanking a transgene has been shown to increase the level and reduce thevariability associated with tansgene expression in plants (Stief et al.,1989; Breyne et al., 1992; Allen et al., 1993; Mlynarova et al., 1994;Spiker and Thompson, 1996). Flanking a transgene or other encoding DNAwith MAR elements may overcome problems associated with differentialbase composition between such transgenes or encoding DNAs andintegrations sites, and/or the detrimental effects of sequences adjacentto transgene integration sites.

The use of enhancers from tissue-specific or developmentally-regulatedgenes may ensure that expression of a linked transgene or other encodingDNA occurs in the appropriately regulated manner.

The use of different combinations of promoters, plastid targetingsequences, and selectable markers for introduced transgenes or otherencoding DNAs can avoid potential problems due to trans-inactivation incases where pyramiding of different transgenes within a single plant isdesired.

Finally, inactivation by co-suppression can be avoided by screening anumber of independent transgenic plants to identify those thatconsistently overexpress particular introduced encoding DNAs (Registeret al., 1994). Site-specific recombination in which the endogenous copyof a gene is replaced by the same gene, but with altered expressioncharacteristics, should obviate this problem (Yoder and Goldsbrough,1994).

Any of the foregoing methods, alone or in combination, can be employedin order to insure the stability of transgene expression in transgenicplants of the present invention.

The invention being thus described, it is obvious that the same can bevaried in many ways. Such variations are not to be regarded as adeparture from the spirit and scope of the present invention, and allsuch modifications as would be obvious to one skilled in the art areintended to be included within the scope of the following claims.

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11 1545 base pairs nucleic acid double linear DNA (genomic) unknown 1ATGGCTGACT CGCAACCCCT GTCCGGTGCT CCGGAAGGTG CCGAATATTT AAGAGCAGTG 60CTGCGCGCGC CGGTTTACGA GGCGGCGCAG GTTACGCCGC TACAAAAAAT GGAAAAACTG 120TCGTCGCGTC TTGATAACGT CATTCTGGTG AAGCGCGAAG ATCGCCAGCC AGTGCACAGC 180TTTAAGCTGC GCGGCGCATA CGCCATGATG GCGGGCCTGA CGGAAGAACA GAAAGCGCAC 240GGCGTGATCA CTGCTTCTGC GGGTAACCAC GCGCAGGGCG TCGCGTTTTC TTCTGCGCGG 300TTAGGCGTGA AGGCCCTGAT CGTTATGCCA ACCGCCACCG CCGACATCAA AGTCGACCGG 360CTGCGCGGCT TCGGCGGCGA AGTGCTGCTC CACGGCGCGA ACTTTGATGA AGCGAAACGC 420AAAGCGATCG AACTGTCACA GCAGCAGGGG TTCACCTGGG TGCCGCCGTT CGACCATCCG 480ATGGTGATTG CCGGGCAAGG CACGCTGGCG CTGGAACTGC TCCAGCAGGA CGCCCATCTC 540GACCGCGTAT TTGTGCCAGT CGGCGGCGGC GGTCTGGCTG CTTGCGTGGC GGTGCTGATC 600AAACAACTGA TGCCGCAAAT CAAAGTGATC GCCGTAGAAG CGGAAGACTC CGCCTGCCTG 660AAAGCAGCGC TGGATGCGGG TCATCCGGTT GATCTGCCGC GCGTAGGGCT ATTTGCTGAA 720GGCGTAGCGG TAAAACGCAT CGGTGACGAA ACCTTCCGTT TATGCCAGGA GTATCTCGAC 780GACATCATCA CCGTCGATAG CGATGCGATC TGTGCGGCGA TGAAGGATTT ATTCGAAGAT 840GTGCGCGCGG TGGCGGAACC CTCTGGCGCG CTGGCGCTGG CGGGAATGAA AAAATATATC 900GCCCTGCACA ACATTCGCGG CGAACGGCTG GCGCATATTC TTTCCGGTGC CAACGTGAAC 960TTCCACGGCC TGCGCTACGT CTCAGAACGC TGCGAACTGG TCGAACAGCG TGAAGCGTTG 1020TTGGCGGTGA CCATTCCGGA AGAAAAAGGC AGCTTCCTCA AATTCTGCCA ACTGCTTGGC 1080GGGCGTTCGG TCACCGAGTT CAACTACCGT TTTGCCGATG CCAAAAACGC CTGCATCTTT 1140GTCGGTGTGC GCCTGAGCCG CGGCCTCGAA GAGCGCAAAG AAATTTTGCA GATGCTCAAC 1200GACGGCGGCT ACAGCGTGGT TGATCTCTCC GACGACGAAA TGGCGAAGCT ACACGTGCGC 1260TATATGGTCG GCGGACGTCC ATCGCATCCG TTGCAGGAAC GCCTCTACAG CTTCGAATTC 1320CCGGAATCAC CGGGCGCGCT GCTGCGCTTC CTCAACACGC TGGGTACGTA CTGGAACATT 1380TCTTTGTTCC ACTATCGCAG CCATGGCACC GACTACGGGC GCGTACTGGC GGCGTTCGAA 1440CTTGGCGACC ATGAACCGGA TTTCGAAACC CGGCTGAATG AGCTGGGCTA CGATTGCCAC 1500GACGAAACCA ATAACCCGGC GTTCAGGTTC TTTTTGGCGG GTTAA 1545 46 base pairsnucleic acid single linear other nucleic acid /desc = “synthetic DNA”unknown 2 TTTTTGGATC CGATATCTTA ACCCGCCAAA AAGAACCTGA ACGCCG 46 37 basepairs nucleic acid single linear other nucleic acid /desc = “syntheticDNA” unknown 3 TTTTTGGATC CATGGCTGAC TCGCAACCCC TGTCCGG 37 44 base pairsnucleic acid single linear other nucleic acid /desc = “synthetic DNA”unknown 4 CAGCTTCGAG TTCCCGGAAT CACCGGGCGC GTTCCTGCGC TTCC 44 1545 basepairs nucleic acid double linear DNA (genomic) unknown 5 ATGGCTGACTCGCAACCCCT GTCCGGTGCT CCGGAAGGTG CCGAATATTT AAGAGCAGTG 60 CTGCGCGCGCCGGTTTACGA GGCGGCGCAG GTTACGCCGC TACAAAAAAT GGAAAAACTG 120 TCGTCGCGTCTTGATAACGT CATTCTGGTG AAGCGCGAAG ATCGCCAGCC AGTGCACAGC 180 TTTAAGCTGCGCGGCGCATA CGCCATGATG GCGGGCCTGA CGGAAGAACA GAAAGCGCAC 240 GGCGTGATCACTGCTTCTGC GGGTAACCAC GCGCAGGGCG TCGCGTTTTC TTCTGCGCGG 300 TTAGGCGTGAAGGCCCTGAT CGTTATGCCA ACCGCCACCG CCGACATCAA AGTCGACCGG 360 CTGCGCGGCTTCGGCGGCGA AGTGCTGCTC CACGGCGCGA ACTTTGATGA AGCGAAACGC 420 AAAGCGATCGAACTGTCACA GCAGCAGGGG TTCACCTGGG TGCCGCCGTT CGACCATCCG 480 ATGGTGATTGCCGGGCAAGG CACGCTGGCG CTGGAACTGC TCCAGCAGGA CGCCCATCTC 540 GACCGCGTATTTGTGCCAGT CGGCGGCGGC GGTCTGGCTG CTTGCGTGGC GGTGCTGATC 600 AAACAACTGATGCCGCAAAT CAAAGTGATC GCCGTAGAAG CGGAAGACTC CGCCTGCCTG 660 AAAGCAGCGCTGGATGCGGG TCATCCGGTT GATCTGCCGC GCGTAGGGCT ATTTGCTGAA 720 GGCGTAGCGGTAAAACGCAT CGGTGACGAA ACCTTCCGTT TATGCCAGGA GTATCTCGAC 780 GACATCATCACCGTCGATAG CGATGCGATC TGTGCGGCGA TGAAGGATTT ATTCGAAGAT 840 GTGCGCGCGGTGGCGGAACC CTCTGGCGCG CTGGCGCTGG CGGGAATGAA AAAATATATC 900 GCCCTGCACAACATTCGCGG CGAACGGCTG GCGCATATTC TTTCCGGTGC CAACGTGAAC 960 TTCCACGGCCTGCGCTACGT CTCAGAACGC TGCGAACTGG TCGAACAGCG TGAAGCGTTG 1020 TTGGCGGTGACCATTCCGGA AGAAAAAGGC AGCTTCCTCA AATTCTGCCA ACTGCTTGGC 1080 GGGCGTTCGGTCACCGAGTT CAACTACCGT TTTGCCGATG CCAAAAACGC CTGCATCTTT 1140 GTCGGTGTGCGCCTGAGCCG CGGCCTCGAA GAGCGCAAAG AAATTTTGCA GATGCTCAAC 1200 GACGGCGGCTACAGCGTGGT TGATCTCTCC GACGACGAAA TGGCGAAGCT ACACGTGCGC 1260 TATATGGTCGGCGGACGTCC ATCGCATCCG TTGCAGGAAC GCCTCTACAG CTTCGAGTTC 1320 CCGGAATCACCGGGCGCGTT CCTGCGCTTC CTCAACACGC TGGGTACGTA CTGGAACATT 1380 TCTTTGTTCCACTATCGCAG CCATGGCACC GACTACGGGC GCGTACTGGC GGCGTTCGAA 1440 CTTGGCGACCATGAACCGGA TTTCGAAACC CGGCTGAATG AGCTGGGCTA CGATTGCCAC 1500 GACGAAACCAATAACCCGGC GTTCAGGTTC TTTTTGGCGG GTTAA 1545 65 base pairs nucleic acidsingle linear other nucleic acid /desc = “synthetic DNA” unknown 6TATCGCAGCC ACGGCACCGA CTACGGGCGC GTACTGGCGG CGTTCGAATT TGGCGACCAT 60GAACC 65 1545 base pairs nucleic acid double linear DNA (genomic)unknown 7 ATGGCTGACT CGCAACCCCT GTCCGGTGCT CCGGAAGGTG CCGAATATTTAAGAGCAGTG 60 CTGCGCGCGC CGGTTTACGA GGCGGCGCAG GTTACGCCGC TACAAAAAATGGAAAAACTG 120 TCGTCGCGTC TTGATAACGT CATTCTGGTG AAGCGCGAAG ATCGCCAGCCAGTGCACAGC 180 TTTAAGCTGC GCGGCGCATA CGCCATGATG GCGGGCCTGA CGGAAGAACAGAAAGCGCAC 240 GGCGTGATCA CTGCTTCTGC GGGTAACCAC GCGCAGGGCG TCGCGTTTTCTTCTGCGCGG 300 TTAGGCGTGA AGGCCCTGAT CGTTATGCCA ACCGCCACCG CCGACATCAAAGTCGACCGG 360 CTGCGCGGCT TCGGCGGCGA AGTGCTGCTC CACGGCGCGA ACTTTGATGAAGCGAAACGC 420 AAAGCGATCG AACTGTCACA GCAGCAGGGG TTCACCTGGG TGCCGCCGTTCGACCATCCG 480 ATGGTGATTG CCGGGCAAGG CACGCTGGCG CTGGAACTGC TCCAGCAGGACGCCCATCTC 540 GACCGCGTAT TTGTGCCAGT CGGCGGCGGC GGTCTGGCTG CTTGCGTGGCGGTGCTGATC 600 AAACAACTGA TGCCGCAAAT CAAAGTGATC GCCGTAGAAG CGGAAGACTCCGCCTGCCTG 660 AAAGCAGCGC TGGATGCGGG TCATCCGGTT GATCTGCCGC GCGTAGGGCTATTTGCTGAA 720 GGCGTAGCGG TAAAACGCAT CGGTGACGAA ACCTTCCGTT TATGCCAGGAGTATCTCGAC 780 GACATCATCA CCGTCGATAG CGATGCGATC TGTGCGGCGA TGAAGGATTTATTCGAAGAT 840 GTGCGCGCGG TGGCGGAACC CTCTGGCGCG CTGGCGCTGG CGGGAATGAAAAAATATATC 900 GCCCTGCACA ACATTCGCGG CGAACGGCTG GCGCATATTC TTTCCGGTGCCAACGTGAAC 960 TTCCACGGCC TGCGCTACGT CTCAGAACGC TGCGAACTGG TCGAACAGCGTGAAGCGTTG 1020 TTGGCGGTGA CCATTCCGGA AGAAAAAGGC AGCTTCCTCA AATTCTGCCAACTGCTTGGC 1080 GGGCGTTCGG TCACCGAGTT CAACTACCGT TTTGCCGATG CCAAAAACGCCTGCATCTTT 1140 GTCGGTGTGC GCCTGAGCCG CGGCCTCGAA GAGCGCAAAG AAATTTTGCAGATGCTCAAC 1200 GACGGCGGCT ACAGCGTGGT TGATCTCTCC GACGACGAAA TGGCGAAGCTACACGTGCGC 1260 TATATGGTCG GCGGACGTCC ATCGCATCCG TTGCAGGAAC GCCTCTACAGCTTCGAATTC 1320 CCGGAATCAC CGGGCGCGCT GCTGCGCTTC CTCAACACGC TGGGTACGTACTGGAACATT 1380 TCTTTGTTCC ACTATCGCAG CCACGGCACC GACTACGGGC GCGTACTGGCGGCGTTCGAA 1440 TTTGGCGACC ATGAACCGGA TTTCGAAACC CGGCTGAATG AGCTGGGCTACGATTGCCAC 1500 GACGAAACCA ATAACCCGGC GTTCAGGTTC TTTTTGGCGG GTTAA 15451545 base pairs nucleic acid double linear DNA (genomic) unknown 8ATGGCTGACT CGCAACCCCT GTCCGGTGCT CCGGAAGGTG CCGAATATTT AAGAGCAGTG 60CTGCGCGCGC CGGTTTACGA GGCGGCGCAG GTTACGCCGC TACAAAAAAT GGAAAAACTG 120TCGTCGCGTC TTGATAACGT CATTCTGGTG AAGCGCGAAG ATCGCCAGCC AGTGCACAGC 180TTTAAGCTGC GCGGCGCATA CGCCATGATG GCGGGCCTGA CGGAAGAACA GAAAGCGCAC 240GGCGTGATCA CTGCTTCTGC GGGTAACCAC GCGCAGGGCG TCGCGTTTTC TTCTGCGCGG 300TTAGGCGTGA AGGCCCTGAT CGTTATGCCA ACCGCCACCG CCGACATCAA AGTCGACCGG 360CTGCGCGGCT TCGGCGGCGA AGTGCTGCTC CACGGCGCGA ACTTTGATGA AGCGAAACGC 420AAAGCGATCG AACTGTCACA GCAGCAGGGG TTCACCTGGG TGCCGCCGTT CGACCATCCG 480ATGGTGATTG CCGGGCAAGG CACGCTGGCG CTGGAACTGC TCCAGCAGGA CGCCCATCTC 540GACCGCGTAT TTGTGCCAGT CGGCGGCGGC GGTCTGGCTG CTTGCGTGGC GGTGCTGATC 600AAACAACTGA TGCCGCAAAT CAAAGTGATC GCCGTAGAAG CGGAAGACTC CGCCTGCCTG 660AAAGCAGCGC TGGATGCGGG TCATCCGGTT GATCTGCCGC GCGTAGGGCT ATTTGCTGAA 720GGCGTAGCGG TAAAACGCAT CGGTGACGAA ACCTTCCGTT TATGCCAGGA GTATCTCGAC 780GACATCATCA CCGTCGATAG CGATGCGATC TGTGCGGCGA TGAAGGATTT ATTCGAAGAT 840GTGCGCGCGG TGGCGGAACC CTCTGGCGCG CTGGCGCTGG CGGGAATGAA AAAATATATC 900GCCCTGCACA ACATTCGCGG CGAACGGCTG GCGCATATTC TTTCCGGTGC CAACGTGAAC 960TTCCACGGCC TGCGCTACGT CTCAGAACGC TGCGAACTGG TCGAACAGCG TGAAGCGTTG 1020TTGGCGGTGA CCATTCCGGA AGAAAAAGGC AGCTTCCTCA AATTCTGCCA ACTGCTTGGC 1080GGGCGTTCGG TCACCGAGTT CAACTACCGT TTTGCCGATG CCAAAAACGC CTGCATCTTT 1140GTCGGTGTGC GCCTGAGCCG CGGCCTCGAA GAGCGCAAAG AAATTTTGCA GATGCTCAAC 1200GACGGCGGCT ACAGCGTGGT TGATCTCTCC GACGACGAAA TGGCGAAGCT ACACGTGCGC 1260TATATGGTCG GCGGACGTCC ATCGCATCCG TTGCAGGAAC GCCTCTACAG CTTCGAGTTC 1320CCGGAATCAC CGGGCGCGTT CCTGCGCTTC CTCAACACGC TGGGTACGTA CTGGAACATT 1380TCTTTGTTCC ACTATCGCAG CCACGGCACC GACTACGGGC GCGTACTGGC GGCGTTCGAA 1440TTTGGCGACC ATGAACCGGA TTTCGAAACC CGGCTGAATG AGCTGGGCTA CGATTGCCAC 1500GACGAAACCA ATAACCCGGC GTTCAGGTTC TTTTTGGCGG GTTAA 1545 1185 base pairsnucleic acid double linear DNA (genomic) unknown 9 ATGACGCGTG AAGTGGTAGTGGTAAGCGGT GTCCGTACCG CGATCGGGAC CTTTGGCGGC 60 AGCCTGAAGG ATGTGGCACCGGCGGAGCTG GGCGCACTGG TGGTGCGCGA GGCGCTGGCG 120 CGCGCGCAGG TGTCGGGCGACGATGTCGGC CACGTGGTAT TCGGCAACGT GATCCAGACC 180 GAGCCGCGCG ACATGTATCTGGGCCGCGTC GCGGCCGTCA ACGGCGGGGT GACGATCAAC 240 GCCCCCGCGC TGACCGTGAACCGCCTGTGC GGCTCGGGCC TGCAGGCCAT TGTCAGCGCC 300 GCGCAGACCA TCCTGCTGGGCGATACCGAC GTCGCCATCG GCGGCGGCGC GGAAAGCATG 360 AGCCGCGCAC CGTACCTGGCGCCGGCAGCG CGCTGGGGCG CACGCATGGG CGACGCCGGC 420 CTGGTCGACA TGATGCTGGGTGCGCTGCAC GATCCCTTCC ATCGCATCCA CATGGGCGTG 480 ACCGCCGAGA ATGTCGCCAAGGAATACGAC ATCTCGCGCG CGCAGCAGGA CGAGGCCGCG 540 CTGGAATCGC ACCGCCGCGCTTCGGCAGCG ATCAAGGCCG GCTACTTCAA GGACCAGATC 600 GTCCCGGTGG TGAGCAAGGGCCGCAAGGGC GACGTGACCT TCGACACCGA CGAGCACGTG 660 CGCCATGACG CCACCATCGACGACATGACC AAGCTCAGGC CGGTCTTCGT CAAGGAAAAC 720 GGCACGGTCA CGGCCGGCAATGCCTCGGGC CTGAACGACG CCGCCGCCGC GGTGGTGATG 780 ATGGAGCGCG CCGAAGCCGAGCGCCGCGGC CTGAAGCCGC TGGCCCGCCT GGTGTCGTAC 840 GGCCATGCCG GCGTGGACCCGAAGGCCATG GGCATCGGCC CGGTGCCGGC GACGAAGATC 900 GCGCTGGAGC GCGCCGGCCTGCAGGTGTCG GACCTGGACG TGATCGAAGC CAACGAAGCC 960 TTTGCCGCAC AGGCGTGCGCCGTGACCAAG GCGCTCGGTC TGGACCCGGC CAAGGTTAAC 1020 CCGAACGGCT CGGGCATCTCGCTGGGCCAC CCGATCGGCG CCACCGGTGC CCTGATCACG 1080 GTGAAGGCGC TGCATGAGCTGAACCGCGTG CAGGGCCGCT ACGCGCTGGT GACGATGTGC 1140 ATCGGCGGCG GGCAGGGCATTGCCGCCATC TTCGAGCGTA TCTGA 1185 15 amino acids amino acid linearprotein unknown 10 Thr Arg Glu Val Val Val Val Ser Gly Val Arg Thr AlaIle Gly 1 5 10 15 394 amino acids amino acid linear protein unknown 11Met Thr Arg Glu Val Val Val Val Ser Gly Val Arg Thr Ala Ile Gly 1 5 1015 Thr Phe Gly Gly Ser Leu Lys Asp Val Ala Pro Ala Glu Leu Gly Ala 20 2530 Leu Val Val Arg Glu Ala Leu Ala Arg Ala Gln Val Ser Gly Asp Asp 35 4045 Val Gly His Val Val Phe Gly Asn Val Ile Gln Thr Glu Pro Arg Asp 50 5560 Met Tyr Leu Gly Arg Val Ala Ala Val Asn Gly Gly Val Thr Ile Asn 65 7075 80 Ala Pro Ala Leu Thr Val Asn Arg Leu Cys Gly Ser Gly Leu Gln Ala 8590 95 Ile Val Ser Ala Ala Gln Thr Ile Leu Leu Gly Asp Thr Asp Val Ala100 105 110 Ile Gly Gly Gly Ala Glu Ser Met Ser Arg Ala Pro Tyr Leu AlaPro 115 120 125 Ala Ala Arg Trp Gly Ala Arg Met Gly Asp Ala Gly Leu ValAsp Met 130 135 140 Met Leu Gly Ala Leu His Asp Pro Phe His Arg Ile HisMet Gly Val 145 150 155 160 Thr Ala Glu Asn Val Ala Lys Glu Tyr Asp IleSer Arg Ala Gln Gln 165 170 175 Asp Glu Ala Ala Leu Glu Ser His Arg ArgAla Ser Ala Ala Ile Lys 180 185 190 Ala Gly Tyr Phe Lys Asp Gln Ile ValPro Val Val Ser Lys Gly Arg 195 200 205 Lys Gly Asp Val Thr Phe Asp ThrAsp Glu His Val Arg His Asp Ala 210 215 220 Thr Ile Asp Asp Met Thr LysLeu Arg Pro Val Phe Val Lys Glu Asn 225 230 235 240 Gly Thr Val Thr AlaGly Asn Ala Ser Gly Leu Asn Asp Ala Ala Ala 245 250 255 Ala Val Val MetMet Glu Arg Ala Glu Ala Glu Arg Arg Gly Leu Lys 260 265 270 Pro Leu AlaArg Leu Val Ser Tyr Gly His Ala Gly Val Asp Pro Lys 275 280 285 Ala MetGly Ile Gly Pro Val Pro Ala Thr Lys Ile Ala Leu Glu Arg 290 295 300 AlaGly Leu Gln Val Ser Asp Leu Asp Val Ile Glu Ala Asn Glu Ala 305 310 315320 Phe Ala Ala Gln Ala Cys Ala Val Thr Lys Ala Leu Gly Leu Asp Pro 325330 335 Ala Lys Val Asn Pro Asn Gly Ser Gly Ile Ser Leu Gly His Pro Ile340 345 350 Gly Ala Thr Gly Ala Leu Ile Thr Val Lys Ala Leu His Glu LeuAsn 355 360 365 Arg Val Gln Gly Arg Tyr Ala Leu Val Thr Met Cys Ile GlyGly Gly 370 375 380 Gln Gly Ile Ala Ala Ile Phe Glu Arg Ile 385 390

What is claimed is:
 1. A plant extract comprising a polyhydroxyalkanoatepolymer, wherein the polyhydroxyalkanoate polymer was produced by theplant; and the polyhydroxyalkanoate polymer has a single mode molecularweight distribution.
 2. The plant extract of claim 1, wherein thepolyhydroxyalkanoate polymer has a molecular weight distribution ofbetween about 1 and about
 5. 3. The plant extract of claim 2, whereinthe polyhydroxyalkanoate polymer has a molecular weight distribution ofbetween about 2 and about
 4. 4. The plant extract of claim 3, whereinthe polyhydroxyalkanoate polymer has a molecular weight distribution ofabout 2.5.
 5. The plant extract of claim 3, wherein thepolyhydroxyalkanoate polymer has a molecular weight distribution ofabout 2.1.
 6. The plant extract of claim 1, wherein thepolyhydroxyalkanoate polymer is a polymer of 3-hydroxybutyrate,3-hydroxyhexanoate, 3-hydroxyoctanoate, 3-hydroxydecanoate,3-hydroxydodecanoate, 3-hydroxytetradecanoate, 3-hydroxyhexadecanoate,3-hydroxyoctadecanoate, 3-hydroxyeicosanoate, 3-hydroxydocosanoate, orcopolymers thereof.
 7. The plant extract of claim 1, wherein thepolyhydroxyalkanoate polymer is a homopolymer.
 8. The plant extract ofclaim 7, wherein the homopolymer is poly(3-hydroxybutyrate) orpoly(4-hydroxybutyrate).
 9. The plant extract of claim 1, wherein thepolyhydroxyalkanoate polymer is a copolymer.
 10. The plant extract ofclaim 7, wherein the copolymer ispoly(3-hydroxybutyrate-co-3-hydroxyvalerate),poly(3-hydroxybutyrate-co-4-hydroxybutyrate),poly(3-hydroxybutyrate-co-3-hydroxyhexanoate),poly(4-hydroxybutyrate-co-3-hydroxyhexanoate), orpoly(3-hydroxybutyrate-co-4-hydroxybutyrate-co-3-hydroxyhexanoate). 11.The plant extract of claim 1, wherein the plant is tobacco, wheat,potato, Arabidopsis, corn, soybean, canola, oil seed rape, sunflower,flax, peanut, sugarcane, switchgrass, or alfalfa.
 12. The plant extractof claim 1, wherein the polyhydroxyalkanoate polymer has a numberaverage molecular weight greater than about 100,000.
 13. The plantextract of claim 12, wherein the polyhydroxyalkanoate polymer has anumber average molecular weight greater than about 300,000.
 14. Theplant extract of claim 13, wherein the polyhydroxyalkanoate polymer hasa number average molecular weight greater than about 500,000.
 15. Apolyhydroxyalkanoate polymer prepared by: (a) inserting into a plantcell nucleic acid molecules encoding a polyhydroxyalkanoate synthasepathway; (b) isolating a transformed plant cell; (c) regenerating thetransformed plant cell to form a transformed plant; and (d) selecting atransformed plant which produces a polyhydroxyalkanoate polymer having asingle mode molecular weight distribution.